Methods and compositions for treating huntington&#39;s disease

ABSTRACT

Disclosed herein are methods and compositions for diagnosing, treating or preventing Huntington&#39;s Disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/902,704, filed Nov. 11, 2013, the disclosure of whichis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure is in the fields of gene expression and genomeediting.

BACKGROUND

Huntington's Disease (HD), also known as Huntington's Chorea, is aprogressive disorder of motor, cognitive and psychiatric disturbances.The mean age of onset for this disease is age 35-44 years, although inabout 10% of cases, onset occurs prior to age 21, and the averagelifespan post-diagnosis of the disease is 15-18 years. Prevalence isabout 3 to 7 among 100,000 people of western European descent.

Huntington's Disease is an example of a trinucleotide repeat expansiondisorders were first characterized in the early 1990s (see Di Prosperoand Fischbeck (2005) Nature Reviews Genetics 6:756-765). These disordersinvolve the localized expansion of unstable repeats of sets of threenucleotides and can result in loss of function of the gene in which theexpanded repeat resides, a gain of toxic function, or both.Trinucleotide repeats can be located in any part of the gene, includingnon-coding and coding gene regions. Repeats located within the codingregions typically involve either a repeated glutamine encoding triplet(CAG) or an alanine encoding triplet (CGA). Expanded repeat regionswithin non-coding sequences can lead to aberrant expression of the genewhile expanded repeats within coding regions (also known as codonreiteration disorders) may cause mis-folding and protein aggregation.The exact cause of the pathophysiology associated with the aberrantproteins is often not known. Typically, in the wild-type genes that aresubject to trinucleotide expansion, these regions contain a variablenumber of repeat sequences in the normal population, but in theafflicted populations, the number of repeats can increase from adoubling to a log order increase in the number of repeats. In HD,repeats are inserted within the N terminal coding region of the largecytosolic protein Huntingtin (Htt). Normal Htt alleles contain 15-20 CAGrepeats, while alleles containing 35 or more repeats can be consideredpotentially HD causing alleles and confer risk for developing thedisease. Alleles containing 36-39 repeats are considered incompletelypenetrant, and those individuals harboring those alleles may or may notdevelop the disease (or may develop symptoms later in life) whilealleles containing 40 repeats or more are considered completelypenetrant. In fact, no asymptomatic persons containing HD alleles withthis many repeats have been reported. Those individuals with juvenileonset HD (<21 years of age) are often found to have 60 or more CAGrepeats. In addition to an increase in CAG repeats, it has also beenshown that HD can involve +1 and +2 frameshifts within the repeatsequences such that the region will encode a poly-serine polypeptide(encoded by AGC repeats in the case of a +1 frameshift) track ratherthan poly-glutamine (Davies and Rubinsztein (2006) Journal of MedicalGenetics 43: 893-896).

In HD, the mutant Htt allele is usually inherited from one parent as adominant trait. Any child born of a HD patient has a 50% chance ofdeveloping the disease if the other parent was not afflicted with thedisorder. In some cases, a parent may have an intermediate HD allele andbe asymptomatic while, due to repeat expansion, the child manifests thedisease. In addition, the HD allele can also display a phenomenon knownas anticipation wherein increasing severity or decreasing age of onsetis observed over several generations due to the unstable nature of therepeat region during spermatogenesis.

Furthermore, trinucleotide expansion in Htt leads to neuronal loss inthe medium spiny gamma-aminobutyric acid (GABA) projection neurons inthe striatum, with neuronal loss also occurring in the neocortex. Mediumspiny neurons (MSN) that contain enkephalin and that project to theexternal globus pallidum (in the so-called “indirect pathway”) are moreinvolved than neurons that contain substance P and project to theinternal globus pallidum (in the “direct” pathway), however both typesof MSN are affected. MSNs in HD display transcriptional dysregulationalong with other abnormal alterations (abnormal aggregations andinclusions of htt, bioenergetic defects, neurotophin deficiency,disorders of axonal transport and exictotoxicity). The mechanism for thetranscriptomic effects may be related to changes in activities ofsoluble DNA-binding transcription factors, abnormalities of chromatinbiochemistry and organization, and aggregate-driven nucleartranscription factor sequestration (see Runne et al (2008) J Neurosci28(39):9723-9731).

Other brain areas greatly affected in people with Huntington's diseaseinclude the substantia nigra, cortical layers 3, 5, and 6, the CA1region of the hippocampus, the angular gyrus in the parietal lobe,Purkinje cells of the cerebellum, lateral tuberal nuclei of thehypothalamus, and the centromedialparafascicular complex of the thalamus(Walker (2007) Lancet 369:218-228).

The role of the normal Htt protein is poorly understood, but it may beinvolved in neurogenesis, apoptotic cell death, and vesicle trafficking.In addition, there is evidence that wild-type Htt stimulates theproduction of brain-derived neurotrophic factor (BDNF), a pro-survivalfactor for the striatal neurons. It has been shown that progression ofHD correlates with a decrease in BDNF expression in mouse models of HD(Zuccato et al (2005) Pharmacological Research 52(2): 133-139), and thatdelivery of either BDNF or glial cell line-derived neurotrophic factor(GDNF) via adeno-associated viral (AAV) vector-mediated gene deliverymay protect striatal neurons in mouse models of HD (Kells et al, (2004)Molecular Therapy 9(5): 682-688).

Treatment options for HD are currently very limited. Some potentialmethodologies designed to prevent the toxicities associated with proteinaggregation that occurs through the extended poly-glutamine tract suchas overexpression of chaperonins or induction of the heat shock responsewith the compound geldanamycin have shown a reduction in thesetoxicities in in vitro models. Other treatments target the role ofapoptosis in the clinical manifestations of the disease. For example,slowing of disease symptoms has been shown via blockage of caspaseactivity in animal models in the offspring of a pairing of mice whereone parent contained a HD allele and the other parent had a dominantnegative allele for caspase 1. Additionally, cleavage of mutant HD Httby caspase may play a role in the pathogenicity of the disease.Transgenic mice carrying caspase-6 resistant mutant Htt were found tomaintain normal neuronal function and did not develop striatalneurodegeneration as compared to mice carrying a non-caspase resistantmutant Htt allele (see Graham et al (2006) Cell 125: 1179-1191).Molecules which target members of the apoptotic pathway have also beenshown to have a slowing effect on symptomology. For example, thecompounds zVAD-fmk and minocycline, both of which inhibit caspaseactivity, have been shown to slow disease manifestation in mice. Thedrug remacemide has also been used in small HD human trials because thecompound was thought to prevent the binding of the mutant Htt to theNDMA receptor to prevent the exertion of toxic effects on the nervecell. However, no statistically significant improvements were observedin neuron function in these trials. In addition, the Huntington StudyGroup conducted a randomized, double-blind study using Co-enzyme Q.Although a trend towards slower disease progression among patients thatwere treated with coenzyme Q10 was observed, there was no significantchange in the rate of decline of total functional capacity. (Di Prosperoand Fischbeck, ibid). U.S. Patent Publications 2011/0082093 and20130253040 disclose nucleases targeted to Htt.

Various methods and compositions for targeted cleavage of genomic DNAhave been described. Such targeted cleavage events can be used, forexample, to induce targeted mutagenesis, induce targeted deletions ofcellular DNA sequences, and facilitate targeted recombination at apredetermined chromosomal locus. See, e.g., U.S. Pat. Nos. 8,623,618;8,034,598; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925;8,110,379; 8,409,861; U.S. Patent Publications 20030232410; 20050208489;20050026157; 20060063231; 20080159996; 201000218264; 20120017290;20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 andU.S. application Ser. No. 14/278,903, the disclosures of which areincorporated by reference in their entireties for all purposes. Thesemethods often involve the use of engineered cleavage systems to induce adouble strand break (DSB) or a nick in a target DNA sequence such thatrepair of the break by an error born process such as non-homologous endjoining (NHEJ) or repair using a repair template (homology directedrepair or HDR) can result in the knock out of a gene or the insertion ofa sequence of interest (targeted integration). This technique can alsobe used to introduce site specific changes in the genome sequencethrough use of a donor oligonucleotide, including the introduction ofspecific deletions of genomic regions, or of specific point mutations orlocalized alterations (also known as gene correction). Cleavage canoccur through the use of specific nucleases such as engineered zincfinger nucleases (ZFN), transcription-activator like effector nucleases(TALENs), or using the CRISPR/Cas system with an engineered crRNA/tracrRNA (‘single guide RNA’) to guide specific cleavage. Further, targetednucleases are being developed based on the Argonaute system (e.g., fromT. thermophilus, known as ‘TtAgo’, see Swarts et al (2014) Nature507(7491): 258-261), which also may have the potential for uses ingenome editing and gene therapy.

Engineered fusion proteins have also been developed for modulatingexpression of a targeted gene. Such proteins can be used for example toenhance or repress expression of a desired gene (see e.g. U.S. Pat. Nos.6,534,261; 6,607,882; 6,824,978; 6,933,113; 7,013,219; 7,220,719;8,268,618; 7,985,778; 8,586,526; U.S. Patent Application 20120294838 thedisclosures of which are incorporated by reference in their entiretiesfor all purposes).

Thus, there remains a need for compositions and methods that can drawfrom these promising techniques for the treatment and prevention ofHuntington's Disease.

SUMMARY

Disclosed herein are methods and compositions for treating Huntington'sDisease. In particular, provided herein are methods and compositions formodifying (e.g., modulating expression of) an HD Htt allele so as totreat Huntington Disease. Also provided are methods and compositions forgenerating animal models of Huntington's Disease.

Thus, in one aspect, engineered (non-naturally occurring) DNA bindingdomains (e.g., zinc finger proteins, TAL effector (TALE) proteins orCRISPR/dCas-TF) that modulate expression of a HD allele (e.g., Htt) areprovided. Engineered zinc finger proteins or TALEs are non-naturallyoccurring zinc finger or TALE proteins whose DNA binding domains (e.g.,recognition helices or RVDs) have been altered (e.g., by selectionand/or rational design) to bind to a pre-selected target site. Any ofthe zinc finger proteins described herein may include 1, 2, 3, 4, 5, 6or more zinc fingers, each zinc finger having a recognition helix thatbinds to a target subsite in the selected sequence(s) (e.g., gene(s)).Similarly, any of the TALE proteins described herein may include anynumber of TALE RVDs. In some embodiments, at least one RVD hasnon-specific DNA binding. In some embodiments, at least one recognitionhelix (or RVD) is non-naturally occurring. In certain embodiments, thezinc finger proteins have the recognition helices shown in Tables 1A and1B. In other embodiments, the zinc finger proteins bind to the targetsequences shown in Tables 2A and 2B. In some embodiments, the zincfinger proteins comprise the recognition helices in Table 2C. In certainembodiments, the zinc finger proteins are formulated into apharmaceutical composition, for example, for administration to asubject.

Engineered (non-naturally occurring) CRISPR/Cas systems are alsoprovided that modulate the expression of a HD allele (e.g. Htt) areprovided. The Cas9 nuclease domain can be specifically engineered tolose DNA cleavage activity (“dCAS”), and fused to a functional domaincapable of modulating gene expression (see Perez-Pimera (2013) NatMethod 10(10):973-976) to create a dCas-TF. When the dCas-TF is suppliedwith an Htt-specific guide RNA, the system modulates Htt geneexpression.

In one aspect, repressors (ZFP-TFs, CRISPR/dCas-TF or TALE-TFs) areprovided that bind to sequences entirely or partially outside the CAGrepeat region of Htt. In another aspect, ZFP, Cas or TALE repressors(ZFP-TFs, CRISPR/dCas-TF or TALE-TFs) are provided that bind tosequences within CAG repeat region of Htt. In some embodiments, theseZFP-TFs, CRISPR/dCas or TALE-TFs preferentially bind to expandedtrinucleotide tracts relative to repeat tracts of a wild-type length,thereby achieving preferential repression of the expanded allele. Insome embodiments these ZFP-TFs, CRISPR/dCas-TFs or TALE-TFs includeprotein interaction domains (or “dimerization domains”) that allowmultimerization when bound to DNA. In some embodiments, these ZFP-TFs,CRISPR/dCas-TFs or TALE TFs achieve cooperative DNA binding to therepeat sequence so that the expanded allele is bound more efficiently bya larger number of ZFPs, dCas or TALE proteins than the wild-typeallele, allowing preferential repression of the mutant allele. Thesecooperative binding ZFP-TFs, CRISPR/dCas-TFs or TALE TFs may or may notfurther contain protein interaction domains that allow multimerizationwhen bound to DNA. In some embodiments, ZFP TFs, CRISPR/dCas-TFs or TALETFs form a stable complex of multimers of a given size, and thus arecapable of preferentially interacting with a CAG tract above a certainminimum size, wherein that minimum size is greater than the length of awild-type CAG tract.

In certain embodiments, the ZFPs, CRISPR/dCas-TFs or TALE proteins asdescribed herein (e.g., two-handed, multimerizing, etc.) preferentiallymodify expression of a mutant Htt allele. In some embodiments, the ZFP,CRISPR/dCas-TF or TALE binds specifically to mutant Htt alleles whereinthe expanded tract encodes poly-glutamine, while in other embodiments,the ZFP, CRISPR/dCas-TF or TALE binds specifically to a mutant Httallele wherein the expansion tract encodes poly-serine. Thus, in someembodiments, the ZFP-TF, CRISPR/dCas-TF or TALE-TF modulates both thewild type and mutant forms of the Htt allele. In certain embodiments,the ZFP, CRISPR/dCas-TF or TALE modulates only the wild type Htt allele.In other embodiments, the ZFP, CRISPR/dCas-TF or TALE modulates only themutant form of Htt.

In other embodiments, repressing ZFP-TFs, CRISPR/dCas-TF or TALE-TFs areprovided which preferentially bind to known SNPs associated with theexpanded HD Htt alleles. In this way, the ZFP-TFs, CRISPR/dCas-TF orTALE-TFs are specific for mutant Htt alleles which contain the SNP,allowing for specific repression of the mutant Htt allele. In anotheraspect, ZFP-TFs, CRISPR/dCas-TF or TALE-TFs that specifically activatethe wild-type Htt allele by interacting with SNPs associated withwild-type alleles are provided. In this way, only the wild-type Httallele is activated.

In certain embodiments, the zinc finger proteins (ZFPs), dCas or TALEproteins as described herein can be placed in operative linkage with aregulatory domain (or functional domain) as part of a fusion protein.The functional domain can be, for example, a transcriptional activationdomain, a transcriptional repression domain and/or a nuclease (cleavage)domain. By selecting either an activation domain or repression domainfor fusion with the ZFP, dCas or TALE, such fusion proteins can be usedeither to activate or to repress gene expression. In some embodiments, afusion protein comprising a ZFP, dCas or TALE targeted to a mutant Httas described herein fused to a transcriptional repression domain thatcan be used to down-regulate mutant Htt expression is provided. In someembodiments, a fusion protein comprising a ZFP, dCas or TALE targeted toa wild-type Htt allele fused to a transcription activation domain thatcan up-regulate the wild type Htt allele is provided. In certainembodiments, the activity of the regulatory domain is regulated by anexogenous small molecule or ligand such that interaction with the cell'stranscription machinery will not take place in the absence of theexogenous ligand. Such external ligands control the degree ofinteraction of the ZFP-TF, CRISPR/dCas-TF or TALE-TF with thetranscription machinery. The regulatory domain(s) may be operativelylinked to any portion(s) of one or more of the ZFPs. dCas or TALEs,including between one or more ZFPs, dCas or TALEs, exterior to one ormore ZFPs, dCas or TALEs and any combination thereof. Any of the fusionproteins described herein may be formulated into a pharmaceuticalcomposition.

In some embodiments, the engineered DNA binding domains as describedherein can be placed in operative linkage with nuclease (cleavage)domains as part of a fusion protein. In some embodiments, the nucleasecomprises a Ttago nuclease. In other embodiments, nuclease systems suchas the CRISPR/Cas system may be utilized with a specific single guideRNA to target the nuclease to a target location in the DNA. In certainembodiments, such nucleases and nuclease fusions may be utilized fortargeting mutant Htt alleles in stem cells such as induced pluripotentstem cells (iPSC), human embryonic stem cells (hESC), mesenchymal stemcells (MSC) or neuronal stem cells wherein the activity of the nucleasefusion will result in an Htt allele containing a wild type number of CAGrepeats. In certain embodiments, pharmaceutical compositions comprisingthe modified stem cells are provided.

In yet another aspect, a polynucleotide encoding any of the DNA bindingproteins described herein is provided In another aspect, polynucleotidesencoding a CRIPSR/Cas nuclease and a single guide RNA are provided. Suchpolynucleotides can be administered to a subject in which it isdesirable to treat Huntington's Disease.

In still further aspects, the invention provides methods andcompositions for the generation of specific model systems for the studyof Huntington's Disease. In certain embodiments, provided herein aremodels in which mutant Htt alleles are generated using embryonic stemcells to generate cell and animal lines in which trinucleotide expansiontracts of specific lengths (50, 80, 109 and 180 CAG repeats, forexample) are inserted into a wild-type Htt allele using zinc fingernuclease (ZFN), TALE-nuclease (TALEN), Ttago or CRISPR/Cas nucleasedriven targeted integration. In certain embodiments, the model systemscomprise in vitro cell lines, while in other embodiments, the modelsystems comprise transgenic animals. In any of the animal modelsdescribed herein, the animal may be, for example, a rodent (e.g., rat,mouse), a primate (e.g., non-human primate) or a rabbit. Thus, theinvention also comprises a cell or cell line in which an endogenous generelated to HD is modified, for example as compared to the wild-typesequence of the gene in the cell. The cell or cell lines may beheterozygous or homozygous for the modification. The modifications maycomprise insertions, deletions and/or combinations thereof. In certainembodiments, the gene (e.g., Htt) is modified by a nuclease (e.g., ZFN,TALEN, CRISPR/Cas system, Ttago system, etc.). In certain embodiments,the modification is at or near the nuclease(s) binding and/or cleavagesite(s), for example, within 1-300 (or any value therebetween) basepairs upstream or downstream of the site(s) of cleavage, more preferablywithin 1-100 base pairs (or any value therebetween) of either side ofthe binding and/or cleavage site(s), even more preferably within 1 to 50base pairs (or any value therebetween) on either side of the bindingand/or cleavage site(s).

In other aspects, the invention comprises delivery of a donor nucleicacid to a target cell. The donor may be delivered prior to, after, oralong with the nucleic acid encoding the nuclease(s). The donor nucleicacid may comprise an exogenous sequence (transgene) to be integratedinto the genome of the cell, for example, an endogenous locus. In someembodiments, the donor may comprise a full length gene or fragmentthereof flanked by regions of homology with the targeted cleavage site.In some embodiments, the donor lacks homologous regions and isintegrated into a target locus through homology independent mechanism(i.e. NHEJ). The donor may comprise any nucleic acid sequence, forexample a nucleic acid that, when used as a substrate forhomology-directed repair of the nuclease-induced double-strand break,leads to a donor-specified deletion to be generated at the endogenouschromosomal locus or, alternatively (or in addition to), novel allelicforms of (e.g., point mutations that ablate a transcription factorbinding site) the endogenous locus to be created. In some aspects, thedonor nucleic acid is an oligonucleotide wherein integration leads to agene correction event, or a targeted deletion.

In some embodiments, the polynucleotide encoding the DNA binding proteinis an mRNA. In some aspects, the mRNA may be chemically modified (Seee.g. Kormann et al, (2011) Nature Biotechnology 29(2): 154-157). Inother aspects, the mRNA may comprise an ARCA cap (see U.S. Pat. Nos.7,074,596 and 8,153,773). In further embodiments, the mRNA may comprisea mixture of unmodified and modified nucleotides (see U.S. PatentPublication 2012-0195936).

In yet another aspect, a gene delivery vector comprising any of thepolynucleotides described herein is provided. In certain embodiments,the vector is an adenovirus vector (e.g., an Ad5/F35 vector), alentiviral vector (LV) including integration competent orintegration-defective lentiviral vectors, or an adenovirus associatedviral vector (AAV). In certain embodiments, the AAV vector is an AAV6vector. Thus, also provided herein are adenovirus (Ad) vectors, LV oradenovirus associate viral vectors (AAV) comprising a sequence encodingat least one nuclease (ZFN or TALEN) and/or a donor sequence fortargeted integration into a target gene. In certain embodiments, the Advector is a chimeric Ad vector, for example an Ad5/F35 vector. Incertain embodiments, the lentiviral vector is an integrase-defectivelentiviral vector (IDLV) or an integration competent lentiviral vector.In certain embodiments the vector is pseudo-typed with a VSV-G envelope,or with other envelopes.

In some embodiments, model systems are provided for Huntington's diseasewherein the target alleles (e.g., mutant Htt) are tagged with expressionmarkers. In certain embodiments, the mutant alleles (e.g., mutant Htt)are tagged. In some embodiments, the wild type allele (e.g., wild-typeHtt) is tagged, and in additional embodiments, both wild type and mutantalleles are tagged with separate expression markers. In certainembodiments, the model systems comprise in vitro cell lines, while inother embodiments, the model systems comprise transgenic animals.

Additionally, pharmaceutical compositions comprising the nucleic acidsand/or proteins (e.g., ZFPs, Cas or TALEs or fusion proteins comprisingthe ZFPs, Cas or TALEs) are also provided. For example, certaincompositions include a nucleic acid comprising a sequence that encodesone of the ZFPs, Cas or TALEs described herein operably linked to aregulatory sequence, combined with a pharmaceutically acceptable carrieror diluent, wherein the regulatory sequence allows for expression of thenucleic acid in a cell. In certain embodiments, the ZFPs, CRISPR/Cas orTALEs encoded are specific for a HD Htt allele. In some embodiments,pharmaceutical compositions comprise ZFPs, CRISPR/Cas or TALEs thatmodulate a HD Htt allele and ZFPs, CRISPR/Cas or TALEs that modulate aneurotrophic factor. Protein based compositions include one of moreZFPs. CRISPR/Cas or TALEs as disclosed herein and a pharmaceuticallyacceptable carrier or diluent.

In yet another aspect also provided is an isolated cell comprising anyof the proteins, polynucleotides and/or compositions as describedherein.

In another aspect, provided herein are methods for treating and/orpreventing Huntington's Disease using the methods and compositionsdescribed herein. In some embodiments, the methods involve compositionswhere the polynucleotides and/or proteins may be delivered using a viralvector, a non-viral vector (e.g., plasmid) and/or combinations thereof.In some embodiments, the methods involve compositions comprising stemcell populations comprising a ZFP or TALE, or altered with the ZFNs,TALENs, Ttago or the CRISPR/Cas nuclease system of the invention.

In some embodiments, the methods and compositions of the invention areused to normalize and/or modify the expression of certain genes inmedium spiny neurons. For example, in some embodiments the methodinvolves use of, and/or the composition is, a genetic repressor thatalters the expression of one or more biomarkers related to HD, includingbut not limited to DARPP-32A, PDE10a, Drd1 and/or Drd2, for thetreatment or prevention of a subject with Huntington's disease. In someembodiments, the genetic repressor is a small molecule, nucleic acid ora protein that inhibits the expression of a huntingtin protein. In someembodiments, the genetic repressor specifically binds to the huntingtingene, in its genomic DNA or in a transcript form, such as mRNA. In someembodiments, the genetic repressor is exogenous and/or includes achemical modification and/or a sequence modification that does not occurin nature. In certain embodiments, the genetic repressor is a ZFP, TALEor Cas DNA binding domain that specifically binds to a coding and/ornon-coding portion of huntingin gene relative to binding to other genesin the genome. In certain other embodiments, the genetic repressor is anantisense nucleic acid that includes a sequence that is 100%, or atleast 90%, or at least 80%, or at least 70%, or at least 60% identicalto a sequence complementary to a coding and/or non-coding portion of thehuntingin gene in its transcript form, such as mRNA. The portion of thehuntingtin gene that the genetic repressor specifically binds to can beat least 8, at least 10, at least 12, at least 15, at least 18, at least20, at least 22, at least 25, at least 30, at least 35, and/or at least40 nucleotides or more in length.

In some embodiments described herein, use of the genetic repressor, suchas for treatment of a subject or for testing of a genetic repressor, canresult in increased expression of one or more of DARPP-32A, PDE10a, Drd1and/or Drd2 relative to an untreated control. An untreated control doesnot receive the genetic repressor, for example, an untreated subject oran untreated control cell, such as an MSN. In some aspects, expressionof DARPP-22 is increased in a MSN (for example, increased by 10% ormore, increased by 20% or more, increased by 30% or more, increased by40% or more, increased by 50% or more, increased by 60% or more,increased by 70% or more, increased by 80% or more, increased by 90% ormore, increased by 100% or more, increased by 125% or more, increased by150% or more, increased by 175% or more, and/or increased by 200% ormore, or any value therebetween) relative to an untreated control (e.g.,an untreated subject or an untreated MSN cell). In other aspects, thelevel of PDE10a is increased in a MSN (for example, increased by 10% ormore, increased by 20% or more, increased by 30% or more, increased by40% or more, increased by 50% or more, increased by 60% or more,increased by 70% or more, increased by 80% or more, increased by 90% ormore, increased by 100% or more, increased by 125% or more, increased by150% or more, increased by 175% or more, and/or increased by 200% ormore, or any value therebetween) relative to an untreated control (e.g.,an untreated subject or an untreated MSN cell). In still furtheraspects, the level of a dopamine receptor Drd1 and/or Drd2 is increasedin a MSN (for example, increased by 10% or more, increased by 20% ormore, increased by 30% or more, increased by 40% or more, increased by50% or more, increased by 60% or more, increased by 70% or more,increased by 80% or more, increased by 90% or more, increased by 100% ormore, increased by 125% or more, increased by 150% or more, increased by175% or more, and/or increased by 200% or more, or any valuetherebetween) relative to an untreated control (e.g., an untreatedsubject or an untreated MSN cell). In some aspects, the level of anycombination of DARPP-32A, PDE10a, Drd1 and Drd2 is increased, forexample, two or more are increased where the two can be DARP32A andPDE10a, or DARP32A and Drd1 or any such combination. In other aspects,the level of any three of DARPP-32A, PDE10a, Drd1 and Drd2 is increased,while in other aspects, the level of DARPP-32A, PDE10a, Drd1 and Drd2are all increased relative to an untreated control (e.g., an untreatedsubject or an untreated MSN cell).

In some embodiments, the methods and compositions of the invention areused to normalize clasping behavior in a subject (e.g., animal model)with HD. For example, in some aspects, the use of the genetic repressor,such as for treatment of a subject or for testing of a geneticrepressor, can result in decreased clasping in a treated subject oranimal model relative to an untreated control. In some aspects, themodel is an HD R6/2 mouse model. In some aspects, clasping behavior isdecreased by 10% or more, decreased by 20% or more, decreased by 30% ormore, decreased by 40% or more, decreased by 50% or more, decreased by60% or more, decreased by 70% or more, decreased by 80% or more,decreased by 90% or more, or decreased by 100% or more, or any valuetherebetween. In some aspects, the clasping decrease can be observedwithin 30 seconds of testing an animal that is within 7 weeks, within 8weeks, within 9 weeks, within 10 weeks, within 11 weeks, or within 12weeks of animal birth and/or within 2 weeks, within 3 weeks, within 4weeks, within 5 weeks, within 6 weeks or within 7 weeks of treatmentwith the genetic repressor.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E are schematics depicting wild type and mutant(Huntington's Disease, HD) Huntingtin (Htt) allele and various ZFP-TFsbinding to those alleles. FIG. 1A shows ZFP designs that bind outside ofthe CAG repeat, and therefore are predicted to bind equally to the wildtype allele and the mutant (HD) allele. “KRAB” refers to the KRABrepression domain from the KOX1 gene and “ZFP” refers to the zinc fingerDNA binding protein. “Standard ZFP TF” is a ZFP transcription factorfusion protein in which the zinc finger DNA binding domains are linkedto the KRAB repression domain. FIG. 1B shows ZFP-TFs designed to bindwithin the CAG region. FIG. 1C depicts a “two-handed ZFP TF,” which is aZFP transcription factor in which two clusters of zinc finger domainsare separated by a rigid protein sequences. The functional (repression)domain is depicted exterior to one ZFP in this Figure, but it will beapparent that the functional domain may be between the ZFPs or exteriorto the ZFPs on either end of the protein. FIG. 1D depicts a“multimerizing ZFP TF,” which is a ZFP TF that is capable ofmultimerizing through a multimerization domain (depicted as speckledboxes). FIG. 1E depicts a ZFP-ZFP-KRAB configuration where two zincfinger DNA binding domains are linked by a flexible linker and are alsofused to a KRAB domain. It will be apparent to the skilled artisan thatin all fusion proteins, the functional domain can be on either end ofthe DNA binding domain, and that the DNA binding domain may comprise awide number range of zinc fingers. Also depicted in FIG. 1 as a box withblack diamonds is a functional domain (e.g., activation, repression,cleavage domain). It will be apparent to the skilled artisan that theexemplary models presented in the Figures may apply to TALE TFs as well.

FIGS. 2A to 2E depict the repression of both alleles of Htt by ZFPs asdescribed in FIG. 1A using ZFP TFs that do not bind to the CAG repeatsequences. The ZFP identification numbers as shown in Tables 1A and 1Bare indicated below the bars. FIG. 2A depicts repression of the humanHtt alleles in HEK293 cells using ZFPs targeted to five loci in thehuman gene. A diagram of the human Htt gene is shown and the locationsof ZFP binding sites are shown. For each ZFP group, each bar representsan independent transfection. FIG. 2B depicts a Western blot showing Httprotein levels in HEK293 cells transfected with the GFP control or the18856 ZFP TF repressor (comprising the KRAB repression domain of KOX1),where the NFκB p65 levels (“p65”) were used to confirm equal proteinloading. The Western blot confirms the repression of Htt expression bythe ZFP-TF. FIG. 2C depicts a similar set of data as FIG. 2A for themouse Htt specific ZFP in Neuro2A cells. As in FIG. 2A, a diagram of themouse Htt gene is shown and the locations of ZFP binding sites areindicated. FIGS. 2D and 2E demonstrate the repression of mouse Htt geneexpression (RNA) in immortalized striatal cells, where different dosesof ZFP-TF mRNA were used for transfection. In all cases except FIG. 2B,Htt mRNA levels were measured by real-time RT-PCR and normalized tothose of Actin mRNA.

FIGS. 3A to 3G depict selective repression of mutant Htt by using ZFPsbinding within the CAG repeat region, as illustrated in FIG. 1B. Thismodel illustrates that the longer CAG repeat region in the mutant alleleallows for increased binding of CAG-targeted ZFP repressor molecules.FIG. 3A depicts different repressor activities on the endogenous Httgene (with normal CAG repeat length) by CAG-targeted ZFPs in HEK293cells. FIG. 3B shows repression of luciferase reporters controlled byHtt promoter/exon1 fragments containing CAG repeats of varying lengths,ranging from 10 to 47 CAG repeats. CAG10 (left-most bar for each of thetwo indicated conditions) shows results with 10 CAG repeats; CAG17 (barsecond from the left for each of the two indicated conditions) showsresults with 17 CAG repeats; CAG23 (bar second from the right for eachof the two indicated conditions) shows results with 23 CAG repeats; andCAG47 (right-most bar for each of the two indicated conditions) showsresults with 47 CAG repeats. The schematic above the graph depicts thearrangement of the Htt promoter, exon 1, the CAG repeats and thereporter luciferase gene used in this system. The data demonstrate thatincreasing the number of CAGs leads to a decreased expression from theHtt promoter by a CAG-targeted ZFP. Furthermore, FIG. 3C demonstratesthat, while a relatively weak CAG-targeted ZFP does not repress theluciferase reporter that contains a normal-length CAG repeat as well asa strong CAG repressor, it drives similar repression of a luciferasereporter that contains an expanded CAG repeat as the strong CAG-targetedZFP. at all doses tested. “pRL-Htt-CAG23-intron 1” (left bar of eachpair) corresponds to expression from the wild type allele while“pRL-HttCAG47-intron 1” (right bar of each pair) correlates withexpression from the mutant expanded Htt allele (containing 47 CAGrepeats). FIG. 3D is a graph depicting repression of mutant Htt (111CAG) by CAG-targeted ZFPs in immortalized mouse striatal cells derivedfrom HdH(Q111/Q7) knock-in mice. Wild-type expression is shown in theleft bar of each pair and knock-in expression in the right bar of eachpair. ZFP-TFs comprising the specified ZFP fused to the KRAB repressiondomain were tested using three different concentrations of ZFP mRNA inthe transfections. FIG. 3E depicts mutant Htt repression by CAG-targetedZFPs in a HD patient derived fibroblast line (CAG15/70). In thisfibroblast line, the wild type Htt allele comprises 15 CAG repeats(“099T(CAG15)”, middle bar of each indicated condition) and the mutantexpanded Htt allele comprises 70 CAG repeats (“099C(CAG70)”, right barof each indicated condition). FIG. 3F shows selective repression ofmutant Htt expression in 4 different HD patient derived fibroblast celllines. The numbers above each grouping indicate the number of CAGrepeats on the wild type Htt allele (e.g. 15 or 18) and on the mutantallele (e.g. 70, 67, 45 and 44); where two different doses of ZFP mRNAwere tested. The left-bar of each pair shows wild-type Htt expressionand the right bar of each shows expression of mutant Htt. FIG. 3Gdepicts Htt expression in HD derived patient fibroblasts as assayed byWestern blot analysis in the presence of the ZFP-TFs 30640, 32528 and30657. The slower migrating protein bands are those produced by theexpanded mutant Htt alleles. 32528 binds to the transcription start siteof Htt (TSS) and thus inhibits expression from both alleles, while 30640and 30657 bind to the CAG repeats (CAG).

FIGS. 4A and 4B depict repression of the mutant Htt in a HD patientderived fibroblast line by a panel of ZFPs targeted to the CAG repeat. Arange of RNA concentrations were used from 0.1 ng to 3 μg. In FIGS. 4Aand 4B, the left bar of each indicated conditions shows total Httexpression, the middle bar shows expression of Htt in fibroblasts inwhich the Htt allele comprises 18 CAG repeats (“099T(CAG18)” and themutant expanded Htt allele comprises 45 CAG repeats 099T (CAG45

FIG. 5 shows the effect of CAG-targeted ZFP repressors on the expressionof Htt and other CAG-containing genes in HD patient-derived fibroblasts.The left bar under each indicated condition shows results with 30640;the middle bar under each indicated condition shows results with 30675;and the right bar shows mock transfections.

FIGS. 6A and 6B depict an experiment that examines the genome-widespecificity of three CAG-targeted ZFPs. FIG. 6A depicts qPCR analysis ofHtt repression performed on six biological replicates (six separatetransfections) of HD fibroblasts (CAG18 (middle bars)/CAG45, right bars)using 30640, 30645, or 33074. The four most similar replicates by qPCRwere then selected for microarray analysis, and the data is presented inFIG. 6B.

FIG. 7 depicts Htt repression in CAG17/69 neuronal stem cells (NSC). Thecells were transfected with ZFP mRNA at indicated doses. Left bars undereach of the indicated doses show results in CAG17 cells, middle barsshow results in wild-type cells, right bars show results in CAG69 cells.

FIG. 8 depicts Htt expression in neurons differentiated from HDembryonic stem cells (ESC) (CAG 17/48) treated with ZFP TFs. The cellswere transfected with ZFP mRNA at indicated doses.

FIG. 9 depicts repression of a mutant Htt transgene expression in R6/2mice following treatment with the ZFP TF 30640.

FIGS. 10A through 10D depict ZFPs with multimerization domains thatspecifically targets expanded CAG repeats, as illustrated in FIG. 1D.FIG. 10A shows a single ZFP that have four components: (i) a KOXrepressor domain (oval labeled “repressor”); (ii) an array of 2-6fingers (two shown, small ovals marked “Z”) that binds to (CAG)_(N) or apermutation of this sequence; and (iii) two dimerization domains(rectangles labeled “d1” and “d2”) that interact in an antiparallelconfiguration. These domains allow the ZFP to polymerize within themajor groove of a CAG tract. FIG. 10B shows a sketch of the bindingevent with a multimer of 3 ZFPs. It will be apparent that any number ofmultimers can be used and that the functional domain may be positionedanywhere on one or more of the individual ZFPs and that these diagramsare applicable to TALE-TFs as well. FIG. 10C shows protein sequences ofthe four ZFP monomer scaffolds that are designed to multimerize viainteractions between dimerizing zinc fingers (DZ). Scaffolds are namedDZ1 (SEQ ID NO:180), DZ2 (SEQ ID NO:181), DZ3 (SEQ ID NO:182) and DZ4(SEQ ID NO: 183). Dimerizing zinc finger domains are underlined, whilethe repression domain and nuclear localization sequence are indicated bybold underline and italic text (respectively). FIG. 10D shows proteinsequences of the seven ZFP monomer scaffolds that are designed tomultimerize via interactions between coiled-coils (CC). Scaffolds arenamed CC1 (SEQ ID NO: 184), CC2 (SEQ ID NO: 185), CC3 (SEQ ID NO: 186),CC4 (SEQ ID NO: 187), CC5 (SEQ ID NO: 188), CC6 (SEQ ID NO: 189) and CC7(SEQ ID NO: 190). Coiled-coil sequences are underlined, while therepression domain and nuclear localization sequence are indicated bybold underline and italic text (respectively). The location of the ZFPregion of each scaffold, which will vary between designs, is indicatedby “[ZFP].” The location of the (DNA-binding) ZFP region of eachscaffold, which will vary between designs, is indicated by “[ZFP].”

FIGS. 11A and 11B depict activity of ZFP-TFs with dimerization domains.In FIG. 11A, ZFP-TFs with “coiled coil” (CC) domains were tested withluciferase reporters. pRL-Htt CAG17 (left bar of each pair) stands forrenilla luciferase reporter controlled by human Htt promoter/exon1fragment with 17 CAG; pGL3-Htt-CAG47 (right bar of each pair) stands forfirefly luciferase reporter controlled by human Htt promoter/exon1fragment with 47 CAG repeats. See text in Example 10 for description ofthe various dimerization domains. In FIG. 11B, ZFPs with the dimerizingzinc finger “DZ” domains were tested with the same luciferase reporters,and demonstrates increased repression with some ZFP-TF dimerizationdomains. The left bar in each doublet indicates the expression from the17CAG repeat Htt allele while the right bar indicates expression from 47CAG repeat Htt allele.

FIGS. 12A and 12B depict repression of Htt by ZFP-ZFP-KOX proteins. FIG.12A depicts Htt repression by the single 33088 and 33084 ZFP-TFs, andrepression by the 33088-33088 and 33088-33084 ZFP-ZFP-KOX proteins inwild-type (left bar), CAG18 (middle bar) and CAG45 (right bar) (FIG.12A) HD fibroblasts; FIG. 12B depicts Htt repression by 33088-33088 and33088-33084 ZFP-ZFP-KOX in wild type (left bar), CAG 20 (middle bar) andCAG41 (left bar) HD fibroblasts.

FIGS. 13A to 13E depict activation of mouse Htt. FIG. 13A demonstratesZFP-TF-driven up-regulation of the mouse Htt genes at the RNA level inNeuro2A cells using a ZFP fused to the p65 activation domain. Doublebars indicate duplicate transfections. FIG. 13B depicts a Western blotdemonstrating increased Htt protein production driven by the ZFP. FIG.13C depicts a wild type mouse Htt allele and a “knock in” Htt allelewhere mouse sequence (most of exon1 and part of intron 1, line abovewild-type allele schematic) has been replaced with corresponding humansequence with CAG expansion (line over knock-in allele schematic). FIG.13D depicts the alignment between mouse sequence (SEQ ID NO: 191) thatwas replaced with the corresponding human sequence (SEQ ID NO: 192) suchthat the knock-in allele has sufficient sequence divergence to allowZFPs (shown in A and B) to be designed to bind specifically the mousesequence. FIG. 13E depicts specific activation of the wild type mouseHtt allele in immortalized striatal cells derived from the HdhQ111/Q7knock-in mice. The left-bar shows results in wild-type cells and rightbar shows results in knock-in mutant allele cells.

FIGS. 14A and 14B depict the results of Cel-I mismatch assays(Surveyor™, Transgenomics) following treatment of K562 cells with Httspecific ZFN pairs. The percent NHEJ activity (in-del) for an active ZFNis shown at the bottom of the corresponding lane. “GFP” indicates cellsthat have been transfected with a GFP encoding plasmid. FIG. 14A depictsresults from ZFNs that cleave early Htt exons while FIG. 14B depictsresults from ZFNs that cleave near the stop codon. Inactive ZFN pairswere also observed (lanes not annotated with in-del percentages).

FIG. 15 depicts graphs of the Htt repression results for severalcandidate TALE-TF proteins. The TALE-TFs were tested in HD patientderived fibroblasts (CAG 20/41). The results demonstrate that some ofthe TALE TFs were active in repressing overall Htt expression, whileothers exhibited mutant Htt-preferential repression.

FIG. 16 depicts two photographs displaying clasping behavior (Mangiariniet al (1996) Cell 87:493-605). The test involves picking up the testmouse by its tail, where it is gently pulled backward and upward by theobserver in a smooth motion until the animal is suspended above thesurface by about 12 inches. The animal is then scored for 30 seconds. HDmice engage in ‘clasping’ behavior as is shown in the photo on the rightwhereas normal control mice display the more open behavior shown on theleft.

FIG. 17 is a graphical display of clasping behavior in a mouse model ofHD (R6/2). The mice are suspended for 30 seconds and scored for the typeand length of clasping behavior over the time period. As can beobserved, the ZFP treated mice showed a decrease in clasping behavior atall time-points over a 12 week period.

FIG. 18 is a graphical display in the change of expression of fourbiomarkers for medium spiny neurons derived from brains of R6/2 micetreated with the ZFP. The four biomarkers analyzed, DARPP-32, PDE10a,DRD1 and DRD2, all showed an increase in expression relative to thesignal from brains of R6/2 mice that had been treated with a GFPexpression vector.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for treating Huntington'sdisease (HD). In particular, Htt-modulating transcription factorscomprising zinc finger proteins (ZFP TFs) or TALEs (TALE-TF) and methodsutilizing such proteins are provided for use in treating or preventingHuntington's disease. For example, ZFP-TFs or TALE-TFs which repressexpression of a mutant Htt allele or activate expression of a wild-typeHtt allele are provided. In addition, zinc finger nucleases (ZFNs), TALEnucleases (TALENs), Ttago systems or CRISPR/Cas nuclease systems thatmodify the genomic structure of the genes associated with HD areprovided. For example, ZFNs, TALENs or CRISPR/Cas nuclease systems thatare able to specifically alter portions of a mutant form of Htt areprovided. These include compositions and methods using engineered zincfinger proteins or engineered TALE proteins, i.e., non-naturallyoccurring proteins which bind to a predetermined nucleic acid targetsequence.

Thus, the methods and compositions described herein provide methods fortreatment and prevention of Huntington's Disease, and these methods andcompositions can comprise zinc finger transcription factors or TALEtranscription factors that are capable of modulating target genes aswell as engineered zinc finger and TALE nucleases, Ttago and CRISPR/Casnuclease systems capable of modifying or editing Htt.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Pat. No. 8,586,526.

Zinc finger binding domains or TALE DNA binding domains can be“engineered” to bind to a predetermined nucleotide sequence, for examplevia engineering (altering one or more amino acids) of the recognitionhelix region of a naturally occurring zinc finger protein or byengineering the RVDs of a TALE protein. Therefore, engineered zincfinger proteins or TALEs are proteins that are non-naturally occurring.Non-limiting examples of methods for engineering zinc finger proteins orTALEs are design and selection. A designed zinc finger protein or TALEis a protein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 8,586,526;6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See, e.g.,8,586,526; 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO01/88197, WO 02/099084.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g., Swarts et al, ibid, G. Sheng et al., (2013) Proc. Natl. Acad.Sci. U.S.A. 111, 652). A “TtAgo system” is all the components requiredincluding, for example, guide DNAs for cleavage by a TtAgo enzyme.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to resynthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingeror TALE proteins can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528, 2008/0131962 and2011/0201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

The term “genetic repressor” refers to any molecule that decreases toany extent the expression of one or more genes. Non-limiting examples ofgenes that can be repressed include biomarkers related to HD (e.g.,DARPP-32A, PDE10a, Drd1 and/or Drd2). Such genetic repression can resultin the treatment and/or prevention of Huntington's disease in a subject.The genetic repressor can be any molecule, including, but not limitedto, a small molecule, a nucleic acid and/or a protein (e.g., a proteincomprising a non-naturally occurring DNA-binding domain such as a zincfinger protein, CRISRP/Cas or TALE domain) that inhibits the expressionof the gene (e.g., huntingtin gene). Genetic repressor proteins cancomprise fusion molecules, for example fusion protein transcriptionfactors (e.g., ZFP-TFs, CRISPR/dCas-TFs or TALE-TFs) comprising aDNA-binding domain and a transcriptional regulation domain or nucleases(e.g., ZFNs, TALENs, CRISPR/Cas and/or Ttago nuclease systems)comprising a DNA-binding domain and a nuclease domain. In someembodiments, the genetic repressor specifically binds to the huntingtingene, in its genomic DNA or in a transcript form, such as mRNA.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “multimerization domain”, (also referred to as a “dimerization domain”or “protein interaction domain”) is a domain incorporated at the amino,carboxy or amino and carboxy terminal regions of a ZFP TF or TALE TF.These domains allow for multimerization of multiple ZFP TF or TALE TFunits such that larger tracts of trinucleotide repeat domains becomepreferentially bound by multimerized ZFP TFs or TALE TFs relative toshorter tracts with wild-type numbers of lengths. Examples ofmultimerization domains include leucine zippers. Multimerization domainsmay also be regulated by small molecules wherein the multimerizationdomain assumes a proper conformation to allow for interaction withanother multimerization domain only in the presence of a small moleculeor external ligand. In this way, exogenous ligands can be used toregulate the activity of these domains.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALE protein asdescribed herein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to an activation domain, the ZFP or TALEDNA-binding domain and the activation domain are in operative linkageif, in the fusion polypeptide, the ZFP or TALE DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe activation domain is able to upregulate gene expression. ZFPs fusedto domains capable of regulating gene expression are collectivelyreferred to as “ZFP-TFs” or “zinc finger transcription factors”, whileTALEs fused to domains capable of regulating gene expression arecollectively referred to as “TALE-TFs” or “TALE transcription factors.”When a fusion polypeptide in which a ZFP DNA-binding domain is fused toa cleavage domain (a “ZFN” or “zinc finger nuclease”), the ZFPDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site. When a fusionpolypeptide in which a TALE DNA-binding domain is fused to a cleavagedomain (a “TALEN” or “TALE nuclease”), the TALE DNA-binding domain andthe cleavage domain are in operative linkage if, in the fusionpolypeptide, the TALE DNA-binding domain portion is able to bind itstarget site and/or its binding site, while the cleavage domain is ableto cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

Biomarkers of Huntington's Disease

Medium spiny neurons are highly susceptible to HD. MSN that containenkephalin and that project to the external globus pallidum (in theso-called “indirect pathway”) are more involved than neurons thatcontain substance P and project to the internal globus pallidum (in the“direct” pathway), however both types of MSN are affected. MSNs in HDdisplay abnormal aggregations and inclusions of htt, bioenergeticdefects, neurotophin deficiency, transcriptional dysregulation,disorders of axonal transport and exictotoxicity.

For example, DARPP-32 (Dopamine- and -cAMP-regulated phosphoprotein, Mr32 kDa) is a major target for dopamine and protein kinase A in thestriatum that acts as a dual-function protein depending on the state andlocation of phosphorylation, in that it can act as an inhibitor ofprotein phosphatase 1 (PP-1) when DARP-32 is phosphorylated at Thr34,and as an inhibitor of PKA when phosphorylated at Thr75 (Svenningsson etal, (2004) Annu Rev Pharmacol Toxicol 44:269-96). It is expressed in 97%of MSNs, in several cortical layers and in cerebellar Purkinje cells.However in mouse models of HD, its mRNA expression is down regulated inMSN to a greater extent that in the cortex (see Ehrlich (2012)Neurotherapeutics 9(2): 270-284).

The striatal-specific cyclic nucleotide phophodiesterase PDE10A acts inthe striatum to limit cyclic nucleotide signaling via enzymatichydrolysis. It is a dual substrate PDE that is expressed at high levelsin both the indirect and direct MSNs (see Kleiman et al, 2011 J ofPharma and Exp Thera 336(1):64-78) and is down regulated in HD.

Dopamine signaling in the striatum is regulated in part by the dopaminereceptors and indirect MSNs express the D1 and D2 dopamine receptors(Drd1 and Drd2, respectively). D1 expressing cells of the direct pathwayproject to the substantia nigra pars reticulate, while D2 receptorexpressing MSNs of the indirect pathway project to the medial globuspallidus, and the classical model of basal ganglia functioning suggeststhat the ability of the striatum to select differential action ofdopamine in the control of movement is due to the segregation of the D1and D2 receptors into the two groups of MSNs (see Gerfen et al (1990)Science 250:1429-1432). Thus, expression of both the Drd1 and Drd2 genesis an essential function of MSNs and an imbalance or defect inexpression can lead to the disruption of motor function observed in HD.

Other biomarkers of MSN function in HD that appear to recapitulate thoseobserved in the human HD caudate include decreases in PENK(proenkephalin), RGS4 (regulator of G-protein signaling 4), thetranscription factor NGFi-A, Calcium/calmodulin-dependent 3′,5′-cyclicnucleotide phosphodiesterase 1B PDE1B, and CNR1 (cannabinoid CB₁receptor) (Runne et al, ibid). Additionally, expression of the mutanthtt gene may be directly analyzed as well.

Clasping behavior in mouse models is indicative of the disease. In therelatively aggressive R6/2 mouse model, a human Htt promoter and exon 1fragment that contains an expanded CAG repeat is ectopically expressed(Mangiarini et al. ibid). These mice exhibit HD-like behavioral changesas well as loss of striatal medium spiny neurons (MSNs), which is ahallmark of HD. Age at death is generally between 10 and 13 weeks(although can occur earlier or later), and the mice display aprogressive neurological phenotype. One of the first symptoms is adyskinesia of the limbs when held by the tail. This progresses to analternating clasping together and releasing of the feet until the miceclasp their feet together immediately after being picked up and can nolonger release this posture. Thus, clasping behavior can be scored as ameasure of degree of HD phenotypic behavior.

Phenotypic improvements in HD may be measured by comparing treated anduntreated animals. The models can be evaluated by measuring claspingbehavior and changes in biomarkers following sacrifice. For example,Rodriguez-Lebron et al (2005, Mol Ther 12(4):618-633 measured theefficacy of a shRNA against htt using an analysis of clasping behaviorand change in DARPP-32 and PENK expression.

DNA-Binding Domains

Described herein are compositions comprising a DNA-binding domain thatspecifically bind to a target sequence in any gene comprising atrinucleotide repeat, including, but not limited to, Htt. AnyDNA-binding domain can be used in the compositions and methods disclosedherein.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;2005/0267061, all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In certain embodiments, the DNA binding domain is an engineered zincfinger protein that binds (in a sequence-specific manner) to a targetsite in a Htt gene and modulates expression of Htt. The ZFPs can bindselectively to either a mutant Htt allele or a wild-type Htt sequence.Htt target sites typically include at least one zinc finger but caninclude a plurality of zinc fingers (e.g., 2, 3, 4, 5, 6 or morefingers). Usually, the ZFPs include at least three fingers. Certain ofthe ZFPs include four, five or six fingers, while some ZFPs include 8,9, 10, 11 or 12 fingers. The ZFPs that include three fingers typicallyrecognize a target site that includes 9 or 10 nucleotides; ZFPs thatinclude four fingers typically recognize a target site that includes 12to 14 nucleotides; while ZFPs having six fingers can recognize targetsites that include 18 to 21 nucleotides. The ZFPs can also be fusionproteins that include one or more regulatory domains, which domains canbe transcriptional activation or repression domains. In someembodiments, the fusion protein comprises two ZFP DNA binding domainslinked together. These zinc finger proteins can thus comprise 8, 9, 10,11, 12 or more fingers. In some embodiments, the two DNA binding domainsare linked via an extendable flexible linker such that one DNA bindingdomain comprises 4, 5, or 6 zinc fingers and the second DNA bindingdomain comprises an additional 4, 5, or 5 zinc fingers. In someembodiments, the linker is a standard inter-finger linker such that thefinger array comprises one DNA binding domain comprising 8, 9, 10, 11 or12 or more fingers. In other embodiments, the linker is an atypicallinker such as a flexible linker. The DNA binding domains are fused toat least one regulatory domain and can be thought of as a ‘ZFP-ZFP-TF’architecture. Specific examples of these embodiments can be referred toas “ZFP-ZFP-KOX” which comprises two DNA binding domains linked with aflexible linker and fused to a KOX repressor and “ZFP-KOX-ZFP-KOX” wheretwo ZFP-KOX fusion proteins are fused together via a linker.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, theDNA-binding specificity of homing endonucleases and meganucleases can beengineered to bind non-natural target sites. See, for example, Chevalieret al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

“Two handed” zinc finger proteins are those proteins in which twoclusters of zinc finger DNA binding domains are separated by interveningamino acids so that the two zinc finger domains bind to twodiscontinuous target sites. An example of a two handed type of zincfinger binding protein is SIP 1, where a cluster of four zinc fingers islocated at the amino terminus of the protein and a cluster of threefingers is located at the carboxyl terminus (see Remacle et al, (1999)EMBO Journal 18 (18): 5073-5084). Each cluster of zinc fingers in theseproteins is able to bind to a unique target sequence and the spacingbetween the two target sequences can comprise many nucleotides.Two-handed ZFPs may include a functional domain, for example fused toone or both of the ZFPs. Thus, it will be apparent that the functionaldomain may be attached to the exterior of one or both ZFPs (see, FIG.1C) or may be positioned between the ZFPs (attached to both ZFPs) (see,FIG. 4).

Specific examples of Htt-targeted ZFPs are disclosed in Tables 1A and1B. The first column in this table is an internal reference name(number) for a ZFP and corresponds to the same name in column 1 ofTables 2A and 2B. “F” refers to the finger and the number following “F”refers which zinc finger (e.g., “F1” refers to finger 1).

TABLE 1A Htt-targeted zinc finger proteins SBS Design # F1 F2 F3 F4 F5F6 18856 RSDDLSR RNDNRTK RSDDLTR RSDDRKT RSADLTR QSSDLRR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: 2) NO: 3) NO: 4) NO: 5)NO: 6) 1) 25920 RSAALSR RSDALAR RSDNLSE KRCNLRC QSSDLRR NA (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: 58) 59) 60) 61) 6)25921 WRSCRSA DRSNLSR QRTHLTQ RSAHLSR TSGHLSR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: 62) 9) 53) 46) 43) 25923 RSDDLSRRNDNRTK WRSCRSA RSDNLAR QSGHLSR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: 1) 2) 62) 7) 41) 25922 RSAALSR RSDALARRSDNLSE KRCNLRC QSSDLSR DRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: NO: 58) 59) 60) 61) 31) 13)

TABLE 1B Human and Mouse Htt-targeted zinc finger proteins SBS Design #F1 F2 F3 F4 F5 F6 32468 RSDNLAR WRGDRVK DRSNLSR TSGSLTR ERGTLAR RSDDRKT(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO:7) 8) 9) 10) 11) 4) 32501 RSDALSR DRSHLAR RSDHLSR QSSDLTR TSGNLTRDRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO:NO: NO: 12) 13) 14) 15) 16) 13) 31809 RSDDLSR RNDNRTK RSDDLTR RSDDRKTRSDDLTR QSSDLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 1) 2) 3) 4) 3) 6) 32528 QSGHLQR TSGNLTR QSGDLTR DRSHLARRSDVLST VRSRLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 17) 16) 18) 13) 19) 20) 30580 RSDNLAR WRGDRVK DRSDLSRRSDALAR ERGTLAR RSDDRKT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 7) 8) 22) 59) 11) 4) 30929 DRSTLRQ DRSDLSRQSSTRAR RSDTLSE HRRSRWG NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:NO: NO: NO: NO: 21) 22) 23) 24) 25) 32538 DRSDLSR RRDTLRS RSDHLSTQSAHRIT QSGDLTR DRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 22) 26) 27) 28) 18) 13) 32567 RSDHLSE QNAHRKTQSSDLSR HRSTRNR QSSDLSR HRSTRNR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: NO: 29) 30) 31) 32) 31) 32) 29627 DRSNLSRLRQDLKR DRSHLTR DRSNLTR RSDHLST QSAHRIT (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 9) 33) 34) 35) 27) 28) 29628TSGNLTR LKQMLAV RSDSLSA DRSDLSR RSDALST DRSTRTK (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 16) 36) 37) 22) 38) 39)29631 QSSDLSR DRSALAR QSSDLSR QSGHLSR RSDVLSE TSGHLSR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 31) 40) 31) 41)42) 43) 29632 RSDTLSE KLCNRKC TSGNLTR HRTSLTD RSAHLSR QSGNLAR (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 24) 44)16) 45) 46) 47) 29637 DRSNLSR QSGNLAR DRSNLSR LKHHLTD QSGDLTR YRWLRNN(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO:9) 47) 9) 48) 18) 49) 29638 RSDHLSQ RSAVRKN QSSDLSR QSGDLTR WSTSLRA NA(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: 50) 51) 31)18) 52) 25917 DRSNLSR QRTHLTQ RSSHLSR TSGSLSR TRQNRDT NA (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: 9) 53) 54) 55) 56) 25916DQSTLRN RSAALSR RSDALAR RSDNLSE KRCNLRC NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: 57) 58) 59) 60) 61) 33074 RSDNLSEKRCNLRC QSGDLTR QSGDLTR RSDNLSE KRCNLRC (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 60) 61) 18) 18) 60) 61) 33080QSGDLTR QSGDLTR RSDNLSE KRCNLRC QSGDLTR QSGDLTR (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 18) 18) 60) 61) 18) 18)33084 QSSDLSR HRSTRNR RSDTLSE RRWTLVG NA NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: 31) 32) 24) 64) 33088 QSSDLSR HRSTRNR RSAVLSEQSSDLSR HRSTRNR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO:NO: NO: 31) 32) 148) 31) 32) 30643 RSDNLSE KRCNLRC QSSDLSR QWSTRKRQSSDLSR QWSTRKR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 60) 61) 31) 63) 31) 63) 30648 RSDNLSE KRCNLRC RSDNLSEKRCNLRC RSDNLSE KRCNLRC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 60) 61) 60) 61) 60) 61) 30645 RSDNLSE KRCNLRCQSSDLSR QWSTRKR QSGDLTR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:NO: NO: NO: NO: 60) 61) 31) 63) 18) 30640 QSSDLSR QWSTRKR QSSDLSRQWSTRKR QSGDLTR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO:NO: NO: 31) 63) 31) 63) 18) 30657 RSDTLSE RRWTLVG QSSDLSR HRSTRNRQSSDLSR HRSTRNR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 24) 64) 31) 32) 31) 32) 30642 QSGDLTR QSSDLSR QWSTRKRQSSDLSR QWSTRKR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO:NO: NO: 18) 31) 63) 31) 63) 30646 RSDNLSE KRCNLRC QSGDLTR QSSDLSRQWSTRKR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO:60) 61) 18) 31) 63) 32220 RSDVLSE QSSDLSR HRSTRNR NA NA NA (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: 42) 31) 32) 32210 QSGDLTR QSSDLSR QWSTRKR NANA NA (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: 18) 31) 63) 32215 RSDNLRERSDNLSE KRCNLRC NA NA NA (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: 65) 60) 61)30658 QSSDLSR HRSTRNR QSSDLSR HRSTRNR QSSDLSR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 31) NO: 32) NO: 31) NO: 32) NO: 31) 32218 QSSDLSRQSSDLSR NA NA NA NA (SEQ ID (SEQ ID NO: NO: 31) 31) 32427 ERGTLARTSGSLTR RSDNLAR DPSNRVG RSDDLSK DNSNRIK (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 11) NO: 10) NO: 7) NO: 78) NO: 149) NO: 150) 32653RSDHLSE QSGHLSR RSDDLTR YRWLLRS QSSDLSR RKDALVA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 29) 41) 3) 66) 31) 67)32677 QSGDLTR RRADLSR DRSHLTR DRSHLAR DRSNLSR LAQPRNK (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 18) 68) 34) 13)9) 69) 33560 ERGTLAR QSGSLTR RSDNLAR DDSHRKD RSDDLSK DNSNRIK (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 11) NO: 84) NO: 7) NO: 151)NO: 149) NO: 150) 33583 DRSNLSR HKQHRDA DRSDLSR RRTDLRR RSANLAR DRSHLAR(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 9) NO: 76) NO: 22)NO: 77) NO: 73) NO: 13) 32685 RSDHLSA RSADRTR RSDVLSE TSGHLSR RSDDLTRTSSDRKK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO:NO: NO: 70) 71) 42) 43) 3) 72) 32422 RSANLAR RSDDLTR RSDTLSE HHSARRCERGTLAR DRSNLTR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 73) 3) 24) 74) 11) 35) 32428 RSDVLST DNSSRTR DRSNLSRHKQHRDA DRSDLSR RRTDLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: NO: NO: NO: NO: NO: 19) 75) 9) 76) 22) 77) 32430 RSDVLST VRSRLRRERGTLAR TSGSLTR RSDNLAR DPSNRVG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: NO: NO: NO: NO: NO: 19) 20) 11) 10) 7) 78) 32432 RSDVLSTVRSRLRR ERGTLAR TSGSLTR RSDHLSA RSADLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 19) 20) 11) 10) 70) 79) 32714RSDVLST DNSSRTR ERGTLAR QSGNLAR DRSHLTR RNDDRKK (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 19) 75) 11) 47) 34) 80)32733 DRSNLSR QKVTLAA RSAHLSR TSGNLTR DRSDLSR RRSTLRS (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 9) 81) 46) 16)22) 82) 30901 DRSALSR QSGSLTR QSSDLSR LKWNLRT RSDNLAR LKWDRQT (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO: NO: NO: NO: NO: 83) 84)31) 85) 7) 86) 31952 QSGALAR RSDDLTR DRSALSR RSDHLTQ QSGDLTR WSTSLRA(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 147) NO: 3) NO: 83)NO: 152) NO: 18) NO: 52) 31921 RSDSLLR RSDDLTR QSGDLTR RRDWLPQ DRSNLSRRSDDRKT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 153) NO: 3)NO: 18) NO: 154) NO: 9) NO: 4) 30906 DRSHLSR TSGNLTR QSGDLTR DRSHLARRSDVLST VRSRLRR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: NO:NO: NO: NO: NO: 87) 16) 18) 13) 19) 20)

The sequence and location for the target sites of these proteins aredisclosed in Tables 2A and 2B. Tables 2A and 2B show the targetsequences for the indicated zinc finger proteins. Nucleotides in thetarget site that are contacted by the ZFP recognition helices areindicated in uppercase letters; non-contacted nucleotides indicated inlowercase.

TABLE 2A Target sites on human and mouse Htt SBS # Target Site 18856AcGCTGCGCCGGCGGAGGCGgggccgcg (SEQ ID NO: 88) 25920gcGCTCAGCAGGTGGTGaccttgtggac_ (SEQ ID NO: 103) 25921atGGTGGGAGAGACTGTgaggcggcagc_ (SEQ ID NO: 104) 25923tgGGAGAGacTGTGAGGCGgcagctggg (SEQ ID NO: 105) 25922atGGCGCTCAGCAGGTGGTGaccttgtg_ (SEQ ID NO: 106)

TABLE 2B Target sites on human and mouse Htt SBS # Target Site 32468agCCGGCCGTGGACTCTGAGccgaggtg_ (SEQ ID NO: 89) 32427cgCACTCGcCGCGAGgGTTGCCgggacg_ (SEQ ID NO: 155) 32501gtGGCGATGCGGGGGGCGTGgtgaggta_ (SEQ ID NO: 90) 31809acGCTGCGCCGGCGGAGGCGgggccgcg_ (SEQ ID NO: 88) 32528ccGGGACGGGTCCAaGATGGAcggccgc_ (SEQ ID NO: 91) 30580agCCGGCCGTGGACTCTGAGccgaggtg_ (SEQ ID NO: 89) 30929ccGTCCCGGCAGCCCCCacggcgccttg_ (SEQ ID NO: 92) 30658ctGCTGCTGCTGCTGCTgctggaaggac_ (SEQ ID NO: 108) 32538cgGGTCCAAGATGGACGGCCgctcaggt_ (SEQ ID NO: 93) 32567ctGCTGCTGCTGCTGGAAGGacttgagg_ (SEQ ID NO: 94) 29627tcAGATGGGACGGCGCTGACctggctgg_ (SEQ ID NO: 95) 29628ctGCCATGGACCTGAATGATgggaccca_ (SEQ ID NO: 96) 29631gtGGTCTGGGAGCTGTCGCTgatgggcg_ (SEQ ID NO: 97) 29632ccGAAGGGCCTGATtCAGCTGttacccc_ (SEQ ID NO: 98) 29637aaCTTGCAAGTAACaGAAGACtcatcct_ (SEQ ID NO: 99) 29638ctTGTACAGCTGTGAGGgtgagcataat_ (SEQ ID NO: 100) 25917gcCATGGTGGGAGAGACtgtgaggcggc_ (SEQ ID NO: 101) 25916ctCAGCAGGTGGTGACCttgtggacatt_ (SEQ ID NO: 102) 33074agCAGCAGcaGCAGCAgCAGCAGcagca_ (SEQ ID NO: 157) 33080caGCAGCAgCAGCAGcaGCAGCAgcagc_ (SEQ ID NO: 107) 33084tgCTGCTGctGCTGCTgctgctggaagg_ (SEQ ID NO: 109) 33088ctGCTGCTgCTGctGCTGCTgctggaag_ (SEQ ID NO: 158) 30643caGCAGCAGCAGCAgCAGCAGcagcagc_ (SEQ ID NO: 107) 30648agCAGCAGCAGCAGCAGCAGcagcagca_ (SEQ ID NO: 157) 30645caGCAGCAGCAgCAGCAGcagcagcagc_ (SEQ ID NO: 107) 30640caGCAGCAGCAGCAGCAgcagcagcagc_ (SEQ ID NO: 107) 30657ctGCTGCTGCTGCTgCTGCTGgaaggac_ (SEQ ID NO: 108) 30642caGCAGCAGCAGCAGCAgcagcagcagc_ (SEQ ID NO: 107) 30646caGCAGCAGCAgCAGCAGcagcagcagc_ (SEQ ID NO: 107) 32220ctGCTGCTgCTGctgctgctgctggaagg_ (SEQ ID NO: 109) 32210caGCAGCAGCAgcagcagcagcagcagc_ (SEQ ID NO: 107) 32215agCAGCAGCAGcagcagcagcagcagca (SEQ ID NO: 110) 32218tGCTGCTgctgctgctgctgctggaagg (SEQ ID NO: 111) 32653ggCTGGCTTTTGCGGGAAGGggcggggc (SEQ ID NO: 112) 32677gaATTGACaGGCGGAtGCGTCGtcctct_ (SEQ ID NO: 113) 33560cgCACTCGcCGCGAGgGTTGCCgggacg_ (SEQ ID NO: 155) 33583gcGGCGAGtGCGTCCCGTGACgtcatgc_ (SEQ ID NO: 158) 32685atTCTGCGGGTCTGGCGTGGcctcgtct_ (SEQ ID NO: 114) 32422gtGACGTCATGCCGGCGGAGacgaggcc_ (SEQ ID NO: 115) 32428gtGCGTCCCGTGACGTCATGccggcgga_ (SEQ ID NO: 116) 32430gcCGCGAGgGTTGCCGGGACGggcccaa_ (SEQ ID NO: 117) 32432ccGCGAGGGTTGCCGGGACGggcccaag_ (SEQ ID NO: 118) 32714caTCGGGCagGAAGCCGTCATGgcaacc_ (SEQ ID NO: 119) 32733tcCTGCCCGATGGGACAGACcctgaaga_ (SEQ ID NO: 120) 30901gtACTGAGcAATGCTGTAGTCagcaatc_ (SEQ ID NO: 121) 31952ccTGTCCAgAGGGTCGCGGTAcctccct_ (SEQ ID NO: 159) 31921tgCCGGACCTGGCAGCGGCGgtggtggc_ (SEQ ID NO: 160) 30906ccGGGACGGGTCCAaGATGGAcggccgc_ (SEQ ID NO: 91)

In certain embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector (TALE)DNA binding domain. See, e.g., U.S. Pat. No. 8,586,526, incorporated byreference in its entirety herein. The plant pathogenic bacteria of thegenus Xanthomonas are known to cause many diseases in important cropplants. Pathogenicity of Xanthomonas depends on a conserved type IIIsecretion (T3S) system which injects more than 25 different effectorproteins into the plant cell. Among these injected proteins aretranscription activator-like effectors (TALE) which mimic planttranscriptional activators and manipulate the plant transcriptome (seeKay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TALEs is AvrBs3 from Xanthomonas campestgris pv.Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TALEs contain a centralized domain of tandem repeats,each repeat containing approximately 34 amino acids, which are key tothe DNA binding specificity of these proteins. In addition, they containa nuclear localization sequence and an acidic transcriptional activationdomain (for a review see Schornack S, et al (2006) J Plant Physiol163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 base pairs in the repeat domain of hpx17.However, both gene products have less than 40% sequence identity withAvrBs3 family proteins of Xanthomonas.

Specificity of these TALEs depends on the sequences found in the tandemrepeats. The repeated sequence comprises approximately 102 base pair andthe repeats are typically 91-100% homologous with each other (Bonas etal, ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues at positions 12 and 13 withthe identity of the contiguous nucleotides in the TALE's target sequence(see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al (2009)Science 326:1509-1512). Experimentally, the code for DNA recognition ofthese TALEs has been determined such that an HD sequence at positions 12and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, C, Gor T, NN binds to A or G, and IG binds to T. These DNA binding repeatshave been assembled into proteins with new combinations and numbers ofrepeats, to make artificial transcription factors that are able tointeract with new sequences and activate the expression of anon-endogenous reporter gene in plant cells (Boch et al, ibid).Engineered TAL proteins have been linked to a FokI cleavage half domainto yield a TAL effector domain nuclease fusion (TALEN) exhibitingactivity in a yeast reporter assay (plasmid based target). Christian etal ((2010)<Genetics epub 10.1534/genetics. 110.120717). See, also, U.S.Pat. No. 8,586,526, incorporated by reference in its entirety.

Specific examples of designed dimerization domains to be used with ZFPsor TALE proteins are listed in Table 3. The amino acid sequences of twotypes of domain, coiled-coil (CC) and dimerizing zinc finger (DZ) arelisted.

TABLE 3 Designed dimerization domains Design name Amino acid sequenceDZ1 TKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYEFSSHIVRGEH (SEQ ID NO: 122) TKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYEFSSHIVRGEH(SEQ ID NO: 122) DZ2FKCEHCRILFLDHVMFTIHMGCHGFRDPFKCNMCGEKCDGPVG LFVHMARNAH(SEQ ID NO: 123)TKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYE FSSHIVRGEH(SEQ ID NO: 122)DZ3 FKCEHCRILFLDHVMFTIHMGCHGFRDPFKCNMCGEKCDGPVGLFVHMARNAH(SEQ ID NO: 123) HHCQHCDMYFADNILYTIHMGCHGYENPFECNICGYHSQDRYEFSSHIVRGEH(SEQ ID NO: 124) DZ4HHCQHCDMYFADNILYTIHMGCHSCDDVFKCNMCGEKCDGPVGLFVHMARNAHGEKPTKCVHCGIVFLDEVMYALHMSCHGFRDPFECNICGYHSQDRYEFSSHIVRGEH(SEQ ID NO: 125)FKCEHCRILFLDHVMFTIHMGCHGFRDPFKCNMCGEKCDGPVGLFVHMARNAHGEKPFYCEHCEITFRDVVMYSLHKGYHGFRDPFECNICGYHSQDRYEFSSHIVRGEH(SEQ ID NO: 126) CC1AQLEKELQALEKKLAQLEWENQALEKELAQ(SEQ ID NO: 127)AQLKKKLQANKKELAQLKWKLQALKKKLAQ(SEQ ID NO: 128) CC2EQLEKKLQALEKKLAQLEWKNQALEKKLAQ(SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ(SEQ ID NO: 130) CC3EQLEKKLQALEKKLAQLEWKNQALEK(SEQ ID NO: 131)ELQANKKELAQLKWELQALKKELAQ(SEQ ID NO: 132) CC4EQLEKKLQALEKKLAQLEWKNQA(SEQ ID NO: 133)QANKKELAQLKWELQALKKELAQ(SEQ ID NO: 134) CC5EQLEKKLQALEKKLAQLEWKNQALEKKLAQ(SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ(SEQ ID NO: 130) CC6EQLEKKLQALEKKLAQLEWKNQALEKKLAQ(SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ(SEQ ID NO: 130) CC7EQLEKKLQALEKKLAQLEWKNQALEKKLAQ(SEQ ID NO: 129)ALKKELQANKKELAQLKWELQALKKELAQ(SEQ ID NO: 130)

Fusion Proteins

Fusion proteins comprising DNA-binding proteins (e.g., ZFPs or TALEs) asdescribed herein and a heterologous regulatory (functional) domain (orfunctional fragment thereof) are also provided. Common domains include,e.g., transcription factor domains (activators, repressors,co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun,fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNArepair enzymes and their associated factors and modifiers; DNArearrangement enzymes and their associated factors and modifiers;chromatin associated proteins and their modifiers (e.g. kinases,acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers. U.S. Pat. Nos. 7,888,121 and 8,409,861 for detailsregarding fusions of DNA-binding domains and nuclease cleavage domains,incorporated by reference in their entireties herein

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipelet al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol.Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol.23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci.25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, API, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

It will be clear to those of skill in the art that, in the formation ofa fusion protein (or a nucleic acid encoding same) between a DNA-bindingdomain and a functional domain, either an activation domain or amolecule that interacts with an activation domain is suitable as afunctional domain. Essentially any molecule capable of recruiting anactivating complex and/or activating activity (such as, for example,histone acetylation) to the target gene is useful as an activatingdomain of a fusion protein. Insulator domains, localization domains, andchromatin remodeling proteins such as ISWI-containing domains and/ormethyl binding domain proteins suitable for use as functional domains infusion molecules are described, for example, in U.S. Pat. Nos. 6,919,204and 7,053,264.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al.(1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; andRobertson et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al.(2000) Plant J. 22:19-27.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin). Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

In certain embodiments, the target site bound by the DNA binding domainis present in an accessible region of cellular chromatin. Accessibleregions can be determined as described, for example, in co-ownedInternational Publication WO 01/83732. If the target site is not presentin an accessible region of cellular chromatin, one or more accessibleregions can be generated as described in co-owned WO 01/83793. Inadditional embodiments, the DNA-binding domain of a fusion molecule iscapable of binding to cellular chromatin regardless of whether itstarget site is in an accessible region or not. For example, suchDNA-binding domains are capable of binding to linker DNA and/ornucleosomal DNA. Examples of this type of “pioneer” DNA binding domainare found in certain steroid receptor and in hepatocyte nuclear factor 3(HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al. (1990)Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

The fusion molecule may be formulated with a pharmaceutically acceptablecarrier, as is known to those of skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned WO00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

Additional exemplary functional domains are disclosed, for example, inco-owned U.S. Pat. Nos. 6,534,261 and 6,933,113.

Functional domains that are regulated by exogenous small molecules orligands may also be selected. For example, RheoSwitch® technology may beemployed wherein a functional domain only assumes its activeconformation in the presence of the external RheoChem™ ligand (see forexample US 20090136465). Thus, the ZFP or TALE may be operably linked tothe regulatable functional domain wherein the resultant activity of theZFP-TF or TALE-TF is controlled by the external ligand.

Nucleases

Also described herein are nucleases, such as ZFNs, TALENs, homingendonucleases, CRISPR/Cas and/or Ttago RNA-guided systems, that areuseful for in vivo for genetic modification, including insertions and/ordeletions made following cleavage of a target by the nuclease(s). Incertain embodiments, the fusion protein comprises a DNA-binding bindingdomain and cleavage (nuclease) domain. As such, gene modification can beachieved using a nuclease, for example an engineered nuclease.Engineered nuclease technology is based on the engineering of naturallyoccurring DNA-binding proteins. For example, engineering of homingendonucleases with tailored DNA-binding specificities has beendescribed. (see, Chames et al. (2005) Nucleic Acids Res 33(20):e178;Arnould et al. (2006) J. Mol. Biol. 355:443-458). In addition,engineering of ZFPs has also been described. See, e.g., U.S. Pat. Nos.8,586,526; 6,534,261; 6,607,882; 6,824,978; 6,979,539; 6,933,113;7,163,824; and 7,013,219.

In addition, ZFPs and TALEs have been fused to nuclease domains tocreate ZFNs and TALENs —functional entities that are able to recognizetheir intended nucleic acid target through their engineered (ZFP orTALE) DNA binding domains and cause the DNA to be cut near the ZFP orTALE DNA binding site via the nuclease activity. See, e.g., Kim et al.(1996) Proc Natl Acad Sci USA 93(3): 1156-1160. Nuclease (e.g., ZFNs,TALENs, CRISPR/Cas and/or Ttago systems) have been used for genomemodification in a variety of organisms. U.S. Pat. Nos. 7,888,121;7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S.Patent Publications 20030232410; 20050208489; 20050026157; 20060063231;20080159996; 201000218264; 20120017290; 20110265198; 20130137104;20130122591; 20130177983 and 20130177960 and U.S. application Ser. No.14/278,903.

Thus, the methods and compositions described herein are broadlyapplicable and may involve any nuclease of interest. Non-limitingexamples of nucleases include CRISPR/Cas and/or Ttago systems,meganucleases, TALENs and zinc finger nucleases. The nuclease maycomprise heterologous DNA-binding and cleavage domains (e.g., zincfinger nucleases; TALENs; meganuclease DNA-binding domains withheterologous cleavage domains) or, alternatively, the DNA-binding domainof a naturally-occurring nuclease may be altered to bind to a selectedtarget site (e.g., a meganuclease that has been engineered to bind tosite different than the cognate binding site).

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family andthe HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI,PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences areknown. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG family, have been used to promote site-specificgenome modification in plants, yeast, Drosophila, mammalian cells andmice, but this approach has been limited to the modification of eitherhomologous genes that conserve the meganuclease recognition sequence(Monet et al. (1999), Biochem. Biophysics. Res. Common. 255: 88-93) orto pre-engineered genomes into which a recognition sequence has beenintroduced (Route et al. (1994), Mol. Cell. Biol. 14:8096-106; Chiltonet al. (2003) Plant Physiology 133:956-65; Puchta et al. (1996), Proc.Natl. Acad. Sci. USA 93: 5055-60; Rong et al. (2002), Genes Dev. 16:1568-81; Gouble et al. (2006), J. Gene Med. 8(5):616-622). Accordingly,attempts have been made to engineer meganucleases to exhibit novelbinding specificity at medically or biotechnologically relevant sites(Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Sussman et al.(2004), J. Mol. Biol. 342: 31-41; Epinat et al. (2003), Nucleic AcidsRes. 31: 2952-62; Chevalier et al. (2002) Molec. Cell 10:895-905; Epinatet al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006)Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66;U.S. Patent Publication Nos. 20070117128; 20060206949; 20060153826;20060078552; and 20040002092). In addition, naturally-occurring orengineered DNA-binding domains from meganucleases have also beenoperably linked with a cleavage domain from a heterologous nuclease(e.g., FokI).

In other embodiments, the nuclease is a zinc finger nuclease (ZFN). ZFNscomprise a zinc finger protein that has been engineered to bind to atarget site in a gene of choice and cleavage domain or a cleavagehalf-domain.

As described in detail above, zinc finger binding domains can beengineered to bind to a sequence of choice. See, for example, Beerli etal. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev.Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660;Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al.(2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc fingerbinding domain can have a novel binding specificity, compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of selection.Rational design includes, for example, using databases comprisingtriplet (or quadruplet) nucleotide sequences and individual zinc fingeramino acid sequences, in which each triplet or quadruplet nucleotidesequence is associated with one or more amino acid sequences of zincfingers which bind the particular triplet or quadruplet sequence. See,for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261,incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length (e.g., TGEKP (SEQ ID NO:135), TGGQRP (SEQ ID NO: 136), TGQKP (SEQ ID NO: 137), and/or TGSQKP(SEQ ID NO: 138)). See, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and7,153,949 for exemplary linker sequences 6 or more amino acids inlength. The proteins described herein may include any combination ofsuitable linkers between the individual zinc fingers of the protein.See, also, U.S. Provisional Patent Publication No. 20110287512.

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and archea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer.” Cas9 cleaves the DNA togenerate blunt ends at the DSB at sites specified by a 20-nucleotideguide sequence contained within the crRNA transcript. Cas9 requires boththe crRNA and the tracrRNA for site specific DNA recognition andcleavage. This system has now been engineered such that the crRNA andtracrRNA can be combined into one molecule (the “single guide RNA”), andthe crRNA equivalent portion of the single guide RNA can be engineeredto guide the Cas9 nuclease to target any desired sequence (see Jinek etal (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471,and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system canbe engineered to create a DSB at a desired target in a genome, andrepair of the DSB can be influenced by the use of repair inhibitors tocause an increase in error prone repair.

As noted above, the cleavage (nuclease) domain of the nuclease may beheterologous to the DNA-binding domain, for example a zinc fingerDNA-binding domain and a cleavage domain from a nuclease or ameganuclease DNA-binding domain and cleavage domain from a differentnuclease. Heterologous cleavage domains can be obtained from anyendonuclease or exonuclease. Exemplary endonucleases from which acleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger- or TALE-FokI fusions, two fusion proteins,each comprising a FokI cleavage half-domain, can be used to reconstitutea catalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger- orTALE-FokI fusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Pat. Nos. 8,772,453; 8,623,618; 8,409,861; 8,034,598;7,914,796; and 7,888,121, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of FokI are all targets forinfluencing dimerization of the FokI cleavage half-domains. Exemplaryengineered cleavage half-domains of FokI that form obligate heterodimersinclude a pair in which a first cleavage half-domain includes mutationsat amino acid residues at positions 490 and 538 of FokI and a secondcleavage half-domain includes mutations at amino acid residues 486 and499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Pat. Nos. 7,914,796 and 8,034,598, the disclosures of which areincorporated by reference in their entirety for all purposes. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). U.S. Pat. No. 8,772,453.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. Pat. Nos.8,772,453; 8,623,618; 8,409,861; 8,034,598; 7,914,796; and 7,888,121.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

In some embodiments, the DNA binding domain is an engineered domain froma TAL effector similar to those derived from the plant pathogensXanthomonas (see Boch et al, (2009) Science 326: 1509-1512 and Moscouand Bogdanove, (2009) Science326: 1501) and Ralstonia (see Heuer et al(2007) Applied and Environmental Microbiology 73(13): 4379-4384). Also,see PCT publication WO2010/079430.

Nucleases (e.g., ZFNs or TALENs, etc.) can be screened for activityprior to use, for example in a yeast-based chromosomal system asdescribed in 8,563,314. Expression of the nuclease may be under thecontrol of a constitutive promoter or an inducible promoter, for examplethe galactokinase promoter which is activated (de-repressed) in thepresence of raffinose and/or galactose and repressed in presence ofglucose.

Delivery

The proteins (e.g., ZFPs, TALEs, CRISPR/Cas, Ttago), polynucleotidesencoding same and compositions comprising the proteins and/orpolynucleotides described herein may be delivered to a target cell byany suitable means including, for example, by injection of ZFP-TF,TALE-TF proteins or by use of ZFN or TALEN encoding mRNA. Suitable cellsinclude but not limited to eukaryotic and prokaryotic cells and/or celllines. Non-limiting examples of such cells or cell lines generated fromsuch cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11,CHO-DUKX, CHOK1SV), VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NSO,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6cells as well as insect cells such as Spodoptera fugiperda (Sf), orfungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. Incertain embodiments, the cell line is a CHO-K1, MDCK or HEK293 cellline. Suitable cells also include stem cells such as, by way of example,embryonic stem cells, induced pluripotent stem cells, hematopoietic stemcells, neuronal stem cells and mesenchymal stem cells.

Methods of delivering proteins comprising zinc finger proteins asdescribed herein are described, for example, in U.S. Pat. Nos.6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, thedisclosures of all of which are incorporated by reference herein intheir entireties.

Zinc finger, TALE or CRISPR/Cas proteins as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger, TALE or CRISPR/Cas protein(s). Any vector systems may beused including, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more zinc finger or TALE protein-encoding sequences. Thus, whenone or more ZFPs, TALEs or CRISPR/Cas proteins are introduced into thecell, the sequences encoding the ZFPs, TALEs or CRISPR/Cas proteins maybe carried on the same vector or on different vectors. When multiplevectors are used, each vector may comprise a sequence encoding one ormultiple ZFPs, TALEs or CRISPR/Cas systems.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs, TALEs or CRISPR/Cassystems in cells (e.g., mammalian cells) and target tissues. Suchmethods can also be used to administer nucleic acids encoding ZFPs,TALEs or a CRISPR/Cas system to cells in vitro. In certain embodiments,nucleic acids encoding the ZFPs, TALEs or CRISPR/Cas system areadministered for in vivo or ex vivo gene therapy uses. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Böhm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,naked RNA, artificial virions, and agent-enhanced uptake of DNA.Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can alsobe used for delivery of nucleic acids. In a preferred embodiment, one ormore nucleic acids are delivered as mRNA. Also preferred is the use ofcapped mRNAs to increase translational efficiency and/or mRNA stability.Especially preferred are ARCA (anti-reverse cap analog) caps or variantsthereof. See U.S. Pat. No. 7,074,596 and U.S. Pat. No. 8,153,773,incorporated by reference herein.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc., (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs, TALEs or CRISPR/Cas systems takeadvantage of highly evolved processes for targeting a virus to specificcells in the body and trafficking the viral payload to the nucleus.Viral vectors can be administered directly to patients (in vivo) or theycan be used to treat cells in vitro and the modified cells areadministered to patients (ex vivo). Conventional viral based systems forthe delivery of ZFPs, TALEs or CRISPR/Cas systems include, but are notlimited to, retroviral, lentivirus, adenoviral, adeno-associated,vaccinia and herpes simplex virus vectors for gene transfer. Integrationin the host genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmouse leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 base pair invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6, AAV8AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such asAAV2/8, AAV2/5 and AAV2/6 can also be used in accordance with thepresent invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney mouse leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFP,TALE or CRISPR/Cas system nucleic acid (gene. cDNA or mRNA), andre-infused back into the subject organism (e.g., patient). In apreferred embodiment, one or more nucleic acids are delivered as mRNA.Also preferred is the use of capped mRNAs to increase translationalefficiency and/or mRNA stability. Especially preferred are ARCA(anti-reverse cap analog) caps or variants thereof. See U.S. Pat. Nos.7,074,596 and 8,153,773, incorporated by reference herein in theirentireties. Various cell types suitable for ex vivo transfection arewell known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and lad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Stem cells that have been modified may also be used in some embodiments.For example, neuronal stem cells that have been made resistant toapoptosis may be used as therapeutic compositions where the stem cellsalso contain the ZFP TFs of the invention. Resistance to apoptosis maycome about, for example, by knocking out BAX and/or BAK using BAX- orBAK-specific TALENs or ZFNs (see, U.S. Patent Publication No.20100003756) in the stem cells, or those that are disrupted in acaspase, again using caspase-6 specific ZFNs for example. These cellscan be transfected with the ZFP TFs or TALE TFs that are known toregulate mutant or wild-type Htt.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, W138, V79, B14AF28-G3, BHK, HaK, NSO,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pischia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used.

Applications

The disclosed compositions and methods can be used for any applicationin which it is desired to modulate the Htt allele, including but notlimited to, therapeutic and research applications.

Diseases and conditions which Htt repressing ZFP TFs or TALE TFs can beused as therapeutic agents include, but are not limited to, Huntington'sdisease. Additionally, methods and compositions comprising ZFNs orTALENs specific for mutant alleles of Htt can be used as a therapeuticfor the treatment of Huntington's disease.

ZFP-TFs or TALE TFs that repress a HD Htt allele may also be used inconjunction with ZFP-TFs or TALE-TFs that activate neutrotrophic factorsincluding, but not limited to, GDNF and BDNF. These ZFPs or TALEs (orpolynucleotides encoding these ZFPs or TALEs) may be administeredconcurrently (e.g., in the same pharmaceutical compositions) or may beadministered sequentially in any order.

Methods and compositions for the treatment of Huntington's disease alsoinclude stem cell compositions wherein a mutant copy of the Htt allelewithin the stem cells has been modified to a wild-type Htt allele usinga Htt-specific ZFN or TALEN.

The methods and compositions of the invention are also useful for thedesign and implementation of in vitro and in vivo models, for example,animal models of trinucleotide repeat disorders, which allows for thestudy of these disorders. Non-limiting examples of suitable in vitromodels include cells or cell lines from any organism, includingfibroblasts. Non-limiting examples of suitable animals for use as animalmodels include, invertebrates (C. elegans, drosophila), rodents (e.g.,rat or mouse), primates (e.g., non-human primates).

The compositions described herein (e.g., proteins and/orpolynucleotides), alone or in combination with other suitable components(e.g. liposomes, nanoparticles or other components known in the art),can be made into aerosol formulations (i.e., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like. Formulations suitable for parenteraladministration, such as, for example, by intravenous, intramuscular,intradermal, and subcutaneous routes, include aqueous and non-aqueous,isotonic sterile injection solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. Compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically or intrathecally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to a patient should be sufficient to effect abeneficial therapeutic response in the patient over time. The dose isdetermined by the efficacy and K_(d) of the particular Htt-bindingmolecule employed, the target cell, and the condition of the patient, aswell as the body weight or surface area of the patient to be treated.The size of the dose also is determined by the existence, nature, andextent of any adverse side-effects that accompany the administration ofa particular compound or vector in a particular patient

EXAMPLES Example 1 Design and Construction of Htt-Targeted Zinc FingerProtein Transcription Factors (ZFP-TFs) and ZFNs

Zinc finger proteins targeted to Htt were engineered essentially asdescribed in U.S. Pat. No. 6,534,261. Tables 1A and 1B show therecognition helices of the DNA binding domain of exemplary Htt-targetedZFPs, while Tables 2A and 2B show the target sequences of these ZFPs.

ZFPs with one contiguous array of zinc fingers were designed to targetsites completely within the CAG repeat region (FIG. 1B). Such ZFPs maybind longer, mutant tracts with higher affinity and/or a higher netoccupancy, achieving selective repression of the mutant allele. ZFNswere also designed that targeted sites which lay partially or whollyoutside of the CAG region (FIG. 1A), and which will therefore bind tothe wild type and mutant allele equally, and regulate expression fromboth alleles with similar efficiency. When designing zinc fingerproteins to recognize the CAG region, a set of one- and two-fingermodules can be employed in a ‘mix and match’ combination. Those modulesare shown below in Table 2C.

TABLE 2C Zinc finger recognition helices used in ZFP-TFstargeting CAG repeats SEQ ID NO: Target site F2 F3 (F2 + F3) CAGCAGRSDNLSE KRCNLRC 161 CAGCAG RSDNLSE KPYNLRT 162 CAGCAG RSDNLSE RLWNRKQ163 CAGCAG RSDNLSV RRWNLRA 164 CAGCAG RSDNLSV RKWNRDS 165 CAGCAG RSDNLSENTSPLML 166 CAGCAG RSDNLSE RRYNLVK 167 CTGCTG RSDTLSE RRWTLVG 168 GCAGCAQSSDLSR QWSTRKR 169 GCAGCA RSAHLSR QSGDLTR 170 GCAGCA QSGDLTR QSGDLTR171 GCAGCA QSGDLTR QSSDLRR 172 GCTGCT QSSDLSR QSSDLRR 173 GCTGCT QSSDLSRHRSTRNR 174 AGC MACCRYA none 175 CAG RSANLRE none 176 CAG RNADRKK none177 CTG RSDVLSE none 42 CTG RSAVLSE none 148 GCA QSGDLTR none 18 GCAQSSDLRR none 6 GCA QNATRIK none 178 GCT QSSDLSR none 31 AAG RSDNLRE none65

Multimerizing ZFP TFs are also constructed as described above exceptthat the vector also contains sequences encoding 1 or more proteininteraction domains (also called dimerization or protein interactiondomains) that enable multimerization of the expressed protein along atract of trinucleotide repeats that is operably linked to the sequencesencoding the ZFP TF. See, FIG. 1D and FIG. 10. Table 3 showsdimerization domain designs that are used with ZFPs targeted to the CAGrepeat region. FIG. 10C shows protein sequences of the four ZFP monomerscaffolds that are designed to multimerize via interactions betweendimerizing zinc fingers (DZ), DZ1-DZ4. Designs are based on workdescribed in Mol. Syst. Biol. (2006) 2:2006.2011. FIG. 10D shows proteinsequences of the seven ZFP monomer scaffolds that are designed tomultimerize via interactions between coiled-coils (CC), CC1-CC7. Thedesign of CC#1 is based on the work described in (J. Am. Chem. Soc.(2001), 123:3151-3152), while CC#2, CC#3 and CC#4 are based on (J. Am.Chem. Soc. (2000), 122:5658-5659). CC and DZ domains allow the ZFP topolymerize within the major groove of a CAG tract (depicted in FIG.10B). By choosing a finger array and dimerization domains withappropriate binding properties, efficient binding will occur only to theexpanded CAG tract of a disease allele.

ZFP-TFs were constructed as fusion proteins comprising a nuclearlocalization sequence, the engineered zinc finger DNA-binding domain(Tables 1A and 1B), targeted to the Htt allele, and a KRAB repressiondomain from the human KOX1 protein. See, FIGS. 1A, 1B and 1D. Thedesigned DNA-binding domains contain 3-6 finger modules, recognizing9-18 base pair sequences (Tables 2A and 2B). Nucleotides in the targetsite that are contacted by the ZFP recognition helices are indicated inuppercase letters; non-contacted nucleotides indicated in lowercase.ZFP-ZFP-TF molecules were also constructed where two ZFP DNA bindingdomains were fused with a flexible linker and fused to a KRAB repressiondomain (FIG. 1E). DNA binding domains were chosen from Tables 2A and 2B.

Example 2 Repression of Both Alleles of Htt in Human and Mouse Cells

To repress both alleles of the Htt (non-allele-specific), ZFPs weredesigned to bind to the Htt promoter and exon 1 region, wherein thetarget site was not entirely within the CAG repeat. See, FIG. 1A. Totest the activity of the Htt repressing ZFP TFs, the ZFP TFs weretransfected into human cells and expression of Htt was monitored usingreal-time RT-PCR.

Human HEK293 cells (Graham et al (1977) J Gen Virol 36:59-74) werecultured in DMEM supplemented with 10% FBS and 1e⁵ cells weretransfected with 1 μg of plasmid DNA encoding indicated ZFP-KOX fusionsby Amaxa Nucleofector® following the manufacturer's instructions.

Transfected cells were incubated for 2 days, and the levels ofendogenous human Huntingtin (Htt) and normalization control beta-actin(ACTB) were analyzed by real-time PCR using Hs00918176_m1 and 4352935Eprimers and probes (Applied Biosystems), respectively, according tostandard protocols. Htt levels were expressed as Htt/ACTB ratiosnormalized to that of the mock-transfected samples (set as 1).

As shown in FIG. 2A, Htt-targeted ZFPs repressed Htt expression. Westernblot analyses were done using standard protocols to confirm thereduction in Htt protein level (FIG. 2B); p65 protein was used asloading control.

Mouse Htt-specific ZFP TFs repressors were transiently transfected intoNeuro2A cells (Klebe & Ruddle (1969) J. Cell Biol. 43: 69A) using theLipofectamine® 2000 kit (Invitrogen) according to manufacturer'sprotocols. mHtt and ACTB mRNA levels were measured at 48 hours aftertransfection using ABI Taqman® primer/probe set Mm01213820 ml and4352933E, respectively. mHtt/ACTB ratios for ZFP transfected sampleswere normalized to that of the GFP control (set as 1).

As shown in FIG. 2C, the ZFPs repressed mouse Htt expression. Inaddition, mouse Htt-specific ZFP-TF repressors can repress mouse Htt inimmortalized striatal cells, STHdh(Q111/Q7), derived from Htt knock-inmice (Trettel et al. (2000) Hum. Mol. Genet 9: 2799-2809). See, FIGS. 2Dand 2E. mRNA for indicated ZFPs were generated using the mMessagemMachine kit (Ambion), and 0.1, 0.5 or 2 μg of these mRNAs weretransfected using Amaxa nucleofector as described above. Cells wereharvested 48 hours after transfection for mHtt and ACTB expressionanalysis as described above. More significant repression in the striatalcells compared to that in Neuro2A cells was observed and may be a resultof enhanced transfection efficiency achieved via mRNA transfection instriatal cells.

Example 3 Selective Repression of Mutant Htt in Human and Mouse Cells

To achieve selective repression of the mutant Htt allele, ZFPs weredesigned to bind within the CAG repeat. FIG. 1B shows one type of suchZFPs, with a contiguous array of zinc fingers linked to a repressiondomain (e.g. the KRAB domain from KOX1); these ZFPs can be designed withappropriate affinity such that threshold occupancy required fromtranscriptional repression can only be established on expanded CAGrepeats. FIGS. 1C, 1D and 1E show three other examples of ZFP designthat can allow specific binding to the expanded CAG repeats.

ZFPs designed as illustrated in FIG. 1B were introduced into HEK293cells and Htt expression evaluated. ZFP-encoding constructs weretransfected into HEK293 cells using FugeneHD using standard protocols.Seventy-two hours after transfection total RNA was isolated and thelevels of endogenous human Huntingtin (Htt) relative to internal controlbeta-actin (ACTB) were analyzed by real-time PCR using Hs00918176_m1 and4352935E primers and probes (Applied Biosystems), respectively. Htt/ACTBratios for ZFP transfected samples were normalized to that of the GFPcontrol (set as 1).

As shown in FIG. 3A, ZFP repressors (fused with KRAB repression domain)designed to bind to CAG repeats (FIG. 1B), either to top or bottomstrand, in HEK293 cells, effectively repressed Htt expression. FIG. 3Adepicts repressors of transcription where expression was measure induplicate transfections (separate bars in the Figure) and multiplereal-time PCR assays completed (error bars). Different levels ofrepression by individual ZFPs suggest that they have different affinityto the CAG repeat region. Because Htt alleles in HEK293 cells have 16and 17 CAG, this result also suggests that “weaker” ZFPs, such as 30640,do not repress Htt alleles with wild-type (unexpanded) CAG repeat lengtheffectively.

To test whether ZFPs such as 30640 can repress transcription of Httalleles with expanded CAG repeats, luciferase reporters controlled byHtt promoter/exon 1 fragment that contains different CAG repeat lengthswere constructed. First, the human Htt promoter/exon1 fragment wasamplified from HEK293 genomic DNA using forward primer:

(SEQ ID NO: 139) 5′ GAAGATCTCACTTGGGGTCCTCAGGTCGTGCCGACand reverse primer:

(SEQ ID NO: 140) 5′ GTATCCAAGCTTCAGCTTTTCCAGGGTCGCCTAGGCGGTCT.

The forward primer introduces a BglII site, the reverse primer changesthe first ATG of Htt into TAG and creates an AvrII site, and alsoincludes a HindIII site. The PCR product was digested with BglII andHindIII and ligated to pRL-TK vector (Promega) that was digested withthe same enzymes to generate the construct pRL-Htt. Then the human Httexon fragment (coding sequence minus first ATG) was amplified fromHEK293 genomic DNA or genomic DNA from HD patients with expanded CAGrepeats using forward primer:

(SEQ ID NO: 141) 5′ GCCTAGGCGACCCTGGAAAAGCTGATGAAGGCCand reverse primer: 5′

(SEQ ID NO: 142) 5′ GTATCCAAGCTTGAGCTGCAGCGGGCCCAAACTCACG.

The forward primer introduces an AvrII site, the reverse primerintroduces a HindIII site. The PCR product was digested with AvrII andHindIII and ligated to pRL-Htt vector that was digested with the sameenzymes. Clones with 10, 17, 23 or 47 CAG repeats (pRL-Htt-CAG(x)) wereidentified by sequencing.

pRL-Htt-CAG(x) reporters (300 ng) and pGL3-promoter reporter (100 ng,used as normalization control, Progema) were transfected into HEK293cells with or without 100 ng of ZFP 30640 expression vector. Firefly(pGL reporter) and renilla (pRL reporter) luciferase activities weremeasured 24 hours after transfection. Renilla luciferase levels werenormalized to those of firefly luciferase from the same transfectedsample, and further normalized to the renilla/firefly ratio of the“reporter only” sample.

As shown in FIG. 3B, repression of the luciferase reporters by ZFP-TF30640 increases with the length of the CAG repeat, suggesting that ZFPswith DNA binding affinities similar to that of 30640 can repress Httpromoter activity via an expanded CAG repeat, and the level ofrepression is dependent on the CAG repeat lengths.

FIG. 3C shows a similar experiment as in FIG. 3B, except the “strong”ZFP-TF 30657 was also tested, and both 30640 and 30657 were tested atmultiple doses as indicated. At every dose level, 30640 gave morerepression of the pRL-Htt-CAG47 reporter than the pRL-Htt-CAG23 reporter(CAG repeat length-dependent repression), while 30657 repressed bothreporters to similar levels. On the pRL-Htt-CAG23 reporter, 30640 gaveless repression than 30657 at every dose level, recapitulating thedifference in their activities on the endogenous Htt allele with normalCAG repeat length (HEK293 cells, FIG. 3A); but on the pRL-Htt-CAG47reporter, 30640 and 30657 gave similar repression at every dose level,suggesting that “weaker” ZFPs such as 30640 can efficiently repress Httpromoter through an expanded CAG repeat, most likely because only anexpanded CAG target can allow threshold occupancy required forrepression to be established by such ZFPs.

FIG. 3D shows ZFP-TFs 30640 and 30657 (fused to the KRAB repressiondomain of KOX1) can repress the knock-in Htt allele (CAG111) inimmortalized striatal cells derived from the Hdh(Q111/Q7) knock-in mice,demonstrating the ZFPs such as 30640, which drives CAG repeatlength-dependent repression of luciferase reporters, can also repressexpression from an endogenous Htt allele that has expanded CAG repeat.mRNA for indicated ZFPs were generated using the mMessage mMachine kit(Ambion), and transfected into Hdh(Q111/Q7) cells at indicated dosesusing Amaxa nucleofector. To detect expression from the wild type mouseHtt allele, forward primer CAGGTCCGGCAGAGGAACC (SEQ ID NO: 193) andreverse primer TTCACACGGTCTTrCTTGGTGG (SEQ ID NO: 194) were used in thereal-time RT-PCR; to detect expression from the knock in Htt allele,forward primer GCCCGGCTGTGGCTGA (SEQ ID NO: 195) and reverse primerTTCACACGGTCTTrCTTGGTGG (SEQ ID NO: 196) were used.

FIG. 3E show the result of testing the ZFP-TFs 30640 and 30657 in an HDpatient-derived fibroblast line (GM21756, Coriell) that has 15 and 70CAGs on the normal and mutant Htt allele, respectively. A SNP-basedallele-specific real-time PCR assay was first established to allowspecific detection from the wild type or the mutant Htt allele. Thephasing of the SNP (rs363099 T/C) was determined by Carroll et al. (MolTher. (2011) 19:2178-85); “T” is on the normal allele and “C” is on themutant allele. To detect Htt expression from the mutant allele (099C),cDNA from the fibroblast was amplified by real-time PCR (SsoFastEvaGreen Supermix, Bio-Rad) using forward primer 099C.F(5′AGTTTGGAGGGTTCTC, SEQ ID NO: 143) and reverse primer 099.R5 (5′TCGACTAAAGCAGGATTCAGG, SEQ ID NO: 144); the annealing/extensiontemperature was 55.6° C. To detect Htt expression from the wild typeallele (099T), real-time PCR of the fibroblast cDNA were done usingforward primer 099T.F (5′AGTTGGAGGGTTTCT, SEQ ID NO: 145), reverseprimer 099.R5 and the 3′phosphorylated blocker oligo 099T.BL(5′AGGGTTTCTCCGCTCAGC-3′phos, SEQ ID NO: 146); the annealing/extensiontemperature was 58.3° C. Total human Huntingtin (hHtt, both wild typeand mutant allele) levels and normalization control beta-actin (ACTB)levels were analyzed by real-time PCR using primer/probe Hs00918176_m1and 4352935E (Applied Biosystems), respectively. For the experimentshown in FIG. 3E, mRNA for indicated ZFPs were generated using themMessage mMachine kit (Ambion), 1 μg of mRNA was transfected using Amaxanucleofector as above. Cells were harvested 48 hours after transfection;mRNA levels from the normal (CAG15, 099T), the mutant (CAG70, 099C) Httallele and total Htt (hHtt) were quantified as described above andnormalized to the levels of ACTB; the Htt/ACTB ratios for each samplewas further normalized to that of the mock-transfected sample. The“strong” CAG-targeted ZFP 30657 repressed both alleles, as expected(based on its activity in HEK293 cells FIG. 3A). ZFP 30640, which showedCAG repeat length-dependent repression of the reporters, gave <10%repression of the wild-type allele while repressed the mutantallele >90%. The levels of total Htt in each sample were consistent withthose of wild type (wt) and mutant Htt levels in the same sample.

ZFP-30640 was also tested in a normal fibroblast line as well as otherHD fibroblast lines that contain different CAG repeat length in the Httgene (see FIG. 3F). Htt expression from each allele was detected asdescribed above. No Htt repression was observed in the normal fibroblastline (CAG18/18). In contrast, excellent allelic discrimination wasobserved in the CAG 15/67 and CAG15/70 lines at both a high and low doseof transfected 30640 mRNA; similar results were obtain for the two HDfibroblast lines with intermediate CAG repeat length on the mutantallele (CAG 18/44 and CAG 18/45)—wherein the expanded allele isrepressed by ˜80% at both the high and low doses of 30640, yet the CAG18allele remained unaffected. Taken together, these data indicate thatallele-specific repressors such as 30640 can maintain strong CAG allelelength selectivity in the context of more prevalent disease genotypessuch as CAG18/44 and CAG18/45.

Western blot analysis was used to show that that ZFPs such as 30640selectively down-regulated mutant Htt protein levels in twopatient-derived fibroblast lines, confirming allele-specific regulationthat was shown by qPCR assays (see FIG. 3G). ZFPs were delivered by mRNAtransfection (Amaxa nucleofection) at a 300 ng dose into 4 replicates of1.5e5 cells and pooled prior to plating in 12-well plates. At 48 hours,cells were washed and harvested for protein extract preparation.Approximately 2.5 μg of extract was loaded onto 5% Tris-acetate gels anddetected by MAB2166 (Millipore). Additionally, the same samples wereloaded on a 4-15% Tris-HCl gels (Bio-Rad) and transferred using standardmethods for detection by an anti B-Actin (1:20,000, Sigma) as loadingcontrols. Based on qPCR studies that measure Htt mRNA, 30640 is anallele-specific repressor targeting the CAG repeat; 32528 is biallelicrepressors targeting the transcription start site (TSS), and 30657 isCAG-targeted repressor that repress both Htt alleles at the dose thatwas used. Western blot showed that 30640 specifically reduced the levelsof mutant Htt (upper band) in both HD patient-derived cell lines, while32528 and 30657 repressed both alleles similarly.

Example 4 Additional CAG-Targeted ZFP Designs that Drive Allele SpecificRepression of Htt

FIG. 4A shows the results of testing ZFP-TFs 30640, 30643, 30645 and33074 (all targeted to the CAG repeat and uses the KRAB repressiondomain) in a CAG18/45 HD fibroblast line. Different amounts of ZFP mRNAwere transfected using Amaxa nucleofector as indicated, expression ofthe mutant Htt (right bar), wild type Htt (middle bar) and total Htt(both alleles, left bar) were measured as described above at 24 hoursafter transfection. ZFPs 30640, 30645 and 33074 drive allele-specificrepression over the entire 3 μg-10 ng ZFP mRNA dose range; while 30643appears to repress both alleles significantly at doses that are 30 ng orhigher, and begins to exhibit allele selectivity at the 10 ng dose.

FIG. 4B shows the results of testing ZFP 30643, 30648, 30657 and 30658(all targeted to CAG repeat and uses the KRAB repression domain) in aCAG15/70 HD fibroblast line. Different amounts of ZFP mRNA weretransfected using Amaxa nucleofector as indicated, expression of themutant Htt (right bar), wild type Htt (middle bar) and total Htt (bothalleles, left bar) were measured as described above at 24 hours aftertransfection. Compared to the ZFPs that were tested in the previousfigure (30640, 30645 and 33074), these ZFPs drive mutant Htt-specificrepression at lower doses. These results suggest that depend on ZFPexpression levels that can be achieved in vivo (e.g. in the brains of HDpatients), allele-specific repression of mutant Htt can be achievedusing appropriate ZFP designs.

Example 5 Repression of Alternate CAG-Containing Genes

Using the RNAs isolated in Example 3 (FIG. 3E), repression of other CAGrepeat containing genes was analyzed, and the results are depicted inFIG. 5. The expression levels of the following genes was examined usingreal-time PCR and normalized to that of Actin: Ataxin 2 (“ATXN2”);Dynamin (“DNM1”); F-box only protein 11 (“FBXO11”), nitrate reductase(“NAP”); Origin recognition complex subunit 4 (“ORC4”); phosphokinase(“PHK”); OCT3/4 protein (“POU3”); THAP domain containing, apoptosisassociated protein 2 (“THAPII”); TATA binding protein (“TBP”); andstanniocalcin 1(“STC1”). In addition, the location of the CAG repeatsequence relative to the transcriptional start site (TSS) was noted, andis indicated in FIG. 3F as “TSS@”, where “+” indicates base position ofCAG repeats that are downstream of TSS, and “−” indicates base positionof CAG repeats that are upstream of TSS. Also, the number of CAG repeats(“#CAG”) is indicated for each gene.

The data demonstrate that repression of the mutant expanded Htt alleleby 30640 is highly specific, and only a subset of CAG repeat-containinggenes whose CAG repeats are relatively close to their respectivetranscription start sites maybe repression targets of ZFPs such as30640.

Example 6 Genome-Wide Specificity of Allele-Specific ZFP Repressors ofHtt

HD fibroblasts (CAG18/45) were transfected to study genome-widespecificity of CAG-targeted ZFPs by microarray analysis (FIG. 6). ZFPswere delivered by mRNA transfection (Amaxa nucleofection) at theindicated doses—ZFPs 30640, 30645 and 33074 were transfected insextuplicate at the 75 ng, 15 ng and 15 ng dose, respectively; GFP-KoxmRNA (150 ng) was transfected as a control, and was used as carrier tobring the total amount of transfected mRNA to 150 ng in all samples.Expression from CAG18 (099T, middle bars) and CAG45 (099C, right bars)alleles was measured by allele-specific qPCR reagents at 24 hours aftertransfection as described above where each of the samples (1-6) arebiological replicates (separate transfections). Htt levels werenormalized to those of GAPDH. Mutant allele-specific repression of Httwas observed for all three ZFPs. The four most similar replicates werethen chosen for microarray analysis (Affymetrix HGU133plus2.0) asfollows: GFP replicate 1, 3, 4 and 6; 30640 replicate 2, 3, 5 and 6;30645 replicate 2, 3, 5 and 6; and 33074 replicate 1, 3, 4 and 5 wereused for microarray analysis. Robust Multi-array Average (RMA) was usedto normalize raw signals from each probe set; ZFP-transfected sampleswere compared to GFP-transfected samples using T-test; “change” callswere made on genes (probe sets) with >2 fold difference relative tocontrol samples and T-test P-value<0.05. Based on that criterion, 30640repressed only two genes, stanniocalcin 1 (STC1) and extendedsynaptotagmin-like protein 1 (ESYT1); 30645 and 33074 only repressed onegene each, STC1 and interleukin 17 receptor A (ILR17RA), respectively.Htt was not detected as a repressed (>2-fold repression) gene becausethe Htt probe set on the array detects both wt and mutant Htt mRNA. Thisexperiment demonstrates that mutant Htt-specific ZFPs, when expressed atlevels that drive efficient allele-specific repression of Htt, canoperate with very high specificity genome-wide.

Example 7 Allele Specific Repression in HD Neural Stem Cells (NSCs)

HD iPSC/ESCs were passaged with accutase and cultured on matrigel coatedplates in E8 media (Life Technologies). Neural stem cells were derivedusing StemPro Neural Induction Medium (Life Technologies). Briefly,iPSC/ESCs were seeded into geltrex coated 6 well dish with 200,000cells/well and when 10-20% confluent the medium was changed to StemProNeural Induction Medium. Medium was changed every 2 days and NSCharvested and expanded on day 7. StemPro NSC SFM medium (LifeTechnologies) was used to culture NSCs. HD NSCs(CAG17/69, derived fromCoriell GM23225 iPSC) were transfected with 1.5 or 0.5 μg ZFP mRNA usingnucleofection. Forty-eight hours post transfection cells were harvestedand expression quantified by RT-PCR. Allele-specific detection of Httexpression was performed using a SNP (rs1143646)-based genotyping assay#4351376 (Applied Biosystems). At the ZFP doses that were tested, 30640gave allele-specific repression of mutant Htt, 30643 gave ˜50%repression of wt Htt and ˜90% repression of mutant Htt, and 30648repressed both alleles (FIG. 7); the behavior of these ZFPs isconsistent with that in HD fibroblasts (FIG. 4). The total Htt levels(middle bars) for each sample is consistent with levels of mutant and wtHtt levels.

Example 8 Htt Repression in Differentiated HD Neurons

HD NSCs were passaged with accutase on geltrex coated plates.

Neuron differentiation was induced by changing medium to neuraldifferentiation medium containing StemPRO NSC SFM medium without (bFGFand EGF). Medium was changed every 3-4 days for up to 21 days. Neuronswere derived from NSC (CAG17/48, derived from HD ESCs) by culture inneural differentiation medium. On day 15 post neural induction cellswere transfected with 1.0 or 0.5 μg ZFP mRNA using nucleofection.Forty-eight hours post transfection cells were harvested and geneexpression quantified by RT-PCR. Because this patient line does notcontain a SNP that allows qPCR-based allele-specific detection of wt andmutant Htt, only total Htt levels can be measured. Because we showedthat 30640 and 33074 does not repress the CAG18 or CAG17 allele in HDfibroblasts and NSCs, the levels of total Htt observed in 30640- and33074-treated samples are consistent with allele-specific repression ofthe mutant allele (CAG48). More potent repression by 30643 and 30648 atthe ZFP doses tested is also consistent with the behavior of these ZFPsin HD fibroblasts (FIG. 8).

Example 9 A CAG Targeted Repressor Represses Mutant Htt Transgene inR6/2 Mice

R6/2 mice (which carries a transgene of mutant human Htt exon 1 with˜120 CAG repeat, see Mangiarini et al, (1996) Cell 15:197) receivedstereotactic, bilateral striatal injections of 3e10 vector genome ofrecombinant AAV2/6 encoding either ZFP 30640-KOX or GFP driven by a CMVpromoter. Mice were injected at 5 weeks of age and sacrificed formolecular analysis at 8 weeks of age. Left and right striata weredissected from each hemisphere and snap frozen. To assess repression ofthe mutant Htt transgene, total RNA was extracted from each striatumwith TRIzol Plus (Life Technologies) followed by cDNA synthesis usingHigh Capacity RT (Life Technologies). Subsequently, R6/2 transgeneexpression was measured by qPCR and normalized to the geometric mean ofthree reference genes (Atp5b, Eif4a2, UbC) as previously described byBenn et al. ((2008) Molecular Neurodegeneration: 3, 17). We observedstatistically significant repression (P<0.001) of the mutant Htttransgene in four ZFP-treated striata relative to the four GFP-treatedcontrol striata (FIG. 9). The average R6/2 repression was 64.9% of theGFP-treated controls. Because complete coverage of the striatum was notachieved using a single stereotactic injection and AAV2/6 preferentiallytransduces neuronal cells, the fold of repression observed (˜35%) islikely an underestimate of actual repression in cells that weretransduced with the AAV vector.

Example 10 Selective Repression of Mutant Htt Using ZFPs withDimerization/Multimerization Domains

In order to engineer zinc finger transcription factors to betterdiscriminate between short CAG and long GAG repeats, we sought to bothdecrease the DNA-binding affinity of individual zinc fingertranscription factors and increase the interaction strength betweendifferent copies of fusion protein bound to adjacent subsites within theCAG repeat. In order to decrease the DNA-binding affinity of individualzinc finger transcription factors, we generated zinc finger domains withfewer zinc fingers and/or with amino acid sequences expected to bind DNAwith less than optimal affinity. In order increase the interactionstrength between different copies of fusion protein bound to adjacentsubsites within the CAG repeat, we fused various dimerization domains toour zinc finger transcription factors. The dimerization domains caninteract in a “parallel” fashion and yield “head-to-head” or“tail-to-tail” dimers of fusion proteins that contain them. Onepotential dimerization strategy requires an array of identicalZFP-transcription factor fusions that bind in a “head-to-tail”orientation and thus this strategy requires dimerization domains thatinteract in an “anti-parallel” fashion. See, e.g., McClain et al. (2001)J. Am. Chem. Soc. 123:3151-3152) and dimerizing zinc finger peptides(Giesecke et al. (2006), Molecular Systems Biology 2:2006.2011).

Dimerization constructs CC 1 and CC2 were based on pairs of antiparallelcoiled coils (McClain et al, ibid, Ghosh et al. (2000) J Am Chem Soc122:5658-5659). Dimerization constructs CC3 and CC4 were truncatedversions of CC2 that lack either 4 residues or 7 residues respectively.Dimerization constructs DZ1, DZ2, DZ3, and DZ4 were based on pairs ofdimerizing zinc finger domains (Giesecke et al, ibid). In each case, onemember of the pair was fused to the N-terminus of the zinc finger DNAbinding domain and the other member of the pair was fused to theC-terminus of the zinc finger DNA binding domain. Short linkers rich inglycine and serine residues were used to fuse the dimerization domainsto the zinc finger binding domain. Additional embodiments of theinvention utilize linkers with alternate lengths and/or amino acidcomposition. Linkers with one or more residues removed or with one ormore glycine or serine residues changes to other amino acid residueswill reduce the flexibility of these linkers and may result in improveddiscrimination between long and short CAG repeats.

To achieve selective repression of the mutant Htt allele, ZFPs weredesigned as illustrated in FIG. 1D and FIGS. 10A and 4B. FIGS. 10C and10D show the sequences of the multimerization domains. FIGS. 11A and 11Bdepict the results that experiments designed to measure the ability ofthe ZFP-TFs comprising the CC and DZ domains, respectively to represstheir targets. For these experiments, indicated ZFP constructs (50 ng)were co-transfected with pRL-Htt-CAG17 (200 ng), pGL3-Htt-CAG47 (200 ng)and pVax-SEAP (secreted alkaline phosphatase, 10 ng, used asnormalization control) into HEK293 cells. Luciferase activity andsecreted alkaline phosphatase activities were measured 24 hours aftertransfection. The Renilla luciferase (CAG17)/SEAP and firefly luciferase(CAG47)/SEAP ratios for each sample were normalized to those of thereporter-only samples. The pGL3-Htt-CAG47 reporter was constructed inthe same way as the pRL-Htt-CAG47 reporter (see Example 3), except thepGL-promoter construct (Promega) was used instead of the pRL-TKconstruct.

As shown in FIG. 11A, 3 ZFPs, when tested as one or more CCdomain-containing constructs, enhanced repression of one or bothreporters when compared to constructs with the same ZFP but no CCdomains. FIG. 11B shows that DZ1 and DZ3 domains enhanced repression of32220 on both reporters.

These results suggest that the CC and DZ domains can in general increaseaffinity of multimerized ZFPs and that design of the DNA binding domainand the dimerization domain may yield optimal CAG-repeat lengthdiscrimination.

Example 11 Selective Repression of Mutant Htt with ZFP-ZFP-Kox Designs

ZFP TFs were tested in HD fibroblasts that were of the ZFP-ZFP-KOXdesign. In these experiments, the two ZFP DNA binding domains werelinked together with a flexible linker (LRQKDAARGSAAMAERPFQ, SEQ ID NO:179) and fused to a KOX repression domain. The linker was placed betweenthe conserved histidines and cysteines. The proteins were tested asdescribed above using ZFP mRNA at indicated doses. These results in theCAG18/45 (FIG. 12A) and CAG20/41 (FIG. 12B) HD fibroblast linesdemonstrated that linking less active ZFP DNA binding domains in thisfashion can result in composite ZFPs that drive allele-specificrepression.

Example 12 Activation of Htt in Mouse Cells

ZFPs as described herein were also evaluated for their ability toactivate Htt expression. ZFPs targeted to the +200 to +467 base pairsregion (relative to the transcription start site) of mouse Htt werefused to the transcriptional activation domain of NFκB p65 subunit. Thistargeting region was chosen because this fragment was replaced by thecorresponding sequence (majority of exon 1 and some intron 1 sequence)from human Htt in various knock-in mouse models of HD (Menalled et al.(2003) J. Comp. Neurol 4651:11-26; Wheeler et al. (2000) Hum Mol Genet8:115-122), therefore ZFPs targeted to this region can selectivelyactivate the wild type allele in those animals but not the knock-inallele.

ZFPs were transfected into Neuro2A cells (both Htt alleles are wild typein these cells), mouse Htt and ACTB mRNA levels were measured asdescribed in Example 2 (duplicate transfections and multiple assays).

As shown in FIG. 13, an increase in Htt mRNA levels as compared to amock transfection was detected using both ZFP-TFs. See, FIG. 13A.Increased Htt protein levels were confirmed by Western blot. See, FIG.13B.

The generation of the knock-in Htt allele is illustrated in FIG. 13C;sequence alignment (FIG. 13D) shows divergence between the mousesequence that was replaced and the corresponding human sequence. FIG.13E shows that when such ZFP activators were transfected intoimmortalized striatal cells derived from HdhQ111/Q7 knock-in mice, onlythe wild type Htt was selectively activated.

Example 13 Regulation of Htt Expression In Vivo

To test efficacy of the Htt-specific ZFP TFs in vivo, AAV2 vectorsencoding the ZFPs are produced. These AAV2 based constructs are thendelivered to the brains of mice. For human Htt-specific ZFP TFs, AAVvectors are delivered to R6/2 mice or BAC HD mice (C57B1/6 or FVB/Nstrains) to assess the repression of the human transgene, as well aschange in HD-like phenotypes. For mouse Htt-specific ZFPs (activators orrepressors), AAV vectors are delivered to wild-type mice (C57B1/6 orFVB/N) or human Htt knock-in mice (HdhQ111/Q7, HdhQ140/Q7 or HdhQ175/Q7)to assess the activation or repression of the endogenous mouse Httexpression. For ZFPs that preferentially targeting the CAG-expandedallele, AAV vectors are delivered to R6/2 mice or human Htt knock-inmice (HdhQ11/Q7, HdhQ140/Q7 or HdhQ175/Q7) to examine the selectiverepression of wt vs. expanded Htt allele. Following sacrifice, braintissues are analyzed for Htt expression by Taqman real-time RT-PCR, anddemonstrate that the Htt genes are modulated by ZFP-TFs.

The transgenic mouse line R6/2 is a well-characterized animal model ofHD in which a human Htt promoter and exon 1 fragment that contains anexpanded CAG repeat is ectopically expressed (Mangiarini et al. (1996)Cell 87, 493-506). These mice exhibit HD-like phenotypic behavioralchanges as well as loss of striatal medium spiny neurons (MSNs), whichis a hallmark of HD. Consistent with MSN degeneration, reducedexpression of MSN markers DARPP-32 (Bibb et al. 2000, Proc. Natl. Acad.Sci. 97(12):6809-6814), phosphodiesterase 10a (PDE10a) (Hebb et al. 2004Neuroscience, 123(4):967-81), dopamine receptor D1 (DRD1) and dopaminereceptor D2 (DRD2) (Cha et al. 1998 Proc. Natl. Acad. Sci. 95(11):6480-6485) have been reported in R6/2 mice.

To test the in vivo efficacy of the genetic repressor, ZFP-33074, whichspecifically represses the mutant Htt allele in HD patient-derivedfibroblasts and neurons, AAV6-33074 (i.e., AAV6 comprising the geneticrepressor) was bilaterally delivered into the striatum of 5-week oldR6/2 mice using stereotaxic injection. Each striatum received AAVinjections at two sites, the coordinates for the anterior and posteriorinfusion was A/P+1.4, M/L+/−1.7, D/V−3.5 and A/P+0.2, M/L+/−2.3,D/V−3.2, respectively. The anterior site received 5 μl of AAV vector andthe posterior site received 4 μl of vector. The titer of the AAV6-33074vector was 1×10¹³ vector genome/ml. Control animals received same doseof AAV6-GFP (i.e., AAV6 comprising green fluorescent protein) to thesame locations in the striatum.

R6/2 mice injected with AAV6-33074 or AAV6-GFP were tested weekly forclasping behavior, which is a well-established motor defect exhibited bythese animals (Mangiarini et al. 1996 Cell 87, 493-506). In brief, eachmouse was removed from its home cage and placed onto the lid of thecage. The animal was then gently pulled backward and upward by theobserver in a smooth motion until the animal is suspended above thesurface by about 12 inches. The animal is then scored for 30 seconds. Ifonly forelimb clasp was observed, the animal was given a score of 1. Ifonly hind limb clasp was observed, the animal was given a score of 2. Ifboth hind limb and forelimb clasp were observed, but not at the sametime, the animal was given a score of 3. A full clasp, defined bysimultaneous hind limb and forelimb clasp pulled tightly into the core,was given a score of 4 (see FIG. 16). After the 30-second suspension,the animal is returned to its home cage. For each treatment group, aswell as age-matched wild type littermates, the proportion of animalsthat displayed full clasping (score of 4) at each weekly observation wasdetermined. Compared to AAV6-GFP-treated animals, AAV6-33074-treatedR6/2 mice displayed reduced frequency of clasping between 7 and 12 weeksof age (when the animals were sacrificed for expression analysis); thereduction was statistically significant (chi-square analysis, p<0.05) at9 and 12 weeks of age (FIG. 17). This result demonstrates that thegenetic repressor of mutant Htt, the ZFP-33074, improved claspingbehavior, a well-characterized motor defect in R6/2 mice.

Expression levels of mutant Htt and MSN marker DARPP-32 in the striatumof R6/2 mice were measured by immunohistochemistry. At 7 weeks afterAAV-33074 injection, R6/2 mice were deeply anesthetized withpentobarbital and perfused through the ascending aorta with isotonicsaline followed by 250 mL of ice-cold 4% paraformaldehyde in 0.1Mphosphate buffer, pH 7.4. The brains were removed and postfixedovernight in the same solution and then transferred to 25% sucrosebefore being sectioned on a freezing-stage microtome at 40 m. Sliceswere washed in PBS and blocked for 4 hours at room temperature in 0.3%Triton X-100 20% Normal GoatSerum/PBS (blocking solution). Rabbitanti-DARPP-32 antibody (Cell Signaling), and mouse anti-mutant Httantibody (Millipore) were incubated for 24 hours at 4° C. in theblocking solution. Afterwards, slices were washed in PBS, incubated for2 hours in 0.3% Triton X-100/PBS with Alexa555 donkey anti rabbit (Lifetechnologies) and Alexa488 goat anti mouse (Life technologies). Finally,slices were washed in PBS, incubated 10 minutes with DAPI (Invitrogen)and mounted with glass coverslips for fluorescent microscopy analysis.Reduced mutant Htt staining was observed in regions ofAAV-33074-injected striatum; importantly, increased DARRP-32 stainingwas observed in the same region, suggesting that the genetic repressorof mutant Htt, ZFP-33074 down-regulation reduced MSN degeneration.

Expression levels of mutant Htt and MSN markers were also measured usingreal-time PCR (Taqman) assays. At 7 weeks after AAV vector infusion(AAV-ZFP or AAV-GFP), R6/2 mouse striata were isolated and dissectedinto anterior, middle and posterior sections and snap frozenindividually. Total RNA was isolated from each striatal section usingTRIzolPlus RNA Purification Kit (Life Technologies), and 500 ng of RNAwas used to generate cDNA using the High Capacity cDNA ReverseTranscription Kit (Life Technologies) in a 20-ul reaction. One hundredthof the cDNA was then used in a real-time PCR reaction to determine thelevels of genes of interest (wild type mouse Htt, mutant Htt transgene,DARPP-32, PDE10a, DRD1 and DRD2) and that of normalization genes (Atp5b,Eif4a2, UbC); the normalization genes were chosen based on Benn et al(2008) Molecular Neurodegeneration 3:17. The primer/probe sets for mouseDARPP-32, PDE10a, Dopamine Receptor D1, Dopamine Receptor D2, Atp5b,Eif4a2 and UbC were purchased from IDT; the primer/probe for mouse Httwas purchased from Life Technologies, and the primer/probe for mutantHtt transgene was as described in Benn et al, ibid. The sequences ofprimer/probe sets are listed in Table 3. Real-time PCR assays wereperformed using SsoFast Probes Supermix (Bio-Rad) in 384-well plateformat on CFX-384 real-time PCR instrument (Bio-Rad) as permanufacture's instruction. Expression analysis was done using CFXmanager software (v3.0). In brief, the expression level of each gene ofinterest and normalization gene was assayed in triplicate reactions andquantified using standard curves spanning a 125-fold concentration range(five-fold dilution series). The expression level of each gene ofinterest was normalized to the average levels of the three normalizationgenes from the same sample (gene of interest/normalization gene ratio);this ratio for each sample was then scaled to that of the mean of allthe AAV-GFP-treated samples (set as 1) and plotted. Statisticalsignificance was assessed using a two-tailed Student's T-test. ForDARPP-32, expression was 178 percent of expression in mice receiving theGFP control, whereas PDE10a, Drd1 and Drd2 were 184%, 189%, and 162% ofexpression in the GFP control, respectively. AAV-ZFP-33074-treatedstriata showed ˜60% repression of mutant Htt relative to AAV-GFP-treatedstriata (P<0.001) while the expression of the wild type mouse Htt wasunchanged. ZFP-treated striata also showed statistically significantincrease in all medium spiny neuron (MSN) markers that were examined(DARPP-32, PDE10A, DRD1 and DRD2), suggesting that genetic repressor ofmutant Htt, ZFP-33074, reduced striatal MSN degeneration in R6/2 mice(FIG. 17).

TABLE 3 Taqman primer/probe sets (all sequences 5′ to 3′) DRD1A Probe/6-FAM/TGC CTT CGG /ZEN/AGT CAT CTT CCT CTC A / IBFQ/ (SEQ ID NO: 208)Primer 1 CCA TCC TTA ACC TCT GTG TGA (SEQ ID NO: 209) Primer 2AAG TCC ATG CTA CGC TAA TCA G (SEQ ID NO: 210) DRD2 Probe/6-FAM/AGC ATC CAT /ZEN/TCT CCG CCT GTT CA/ IBFQ/ (SEQ ID NO: 211)Primer 1 TGA CAG CAT CTC CAT TTC CAG (SEQ ID NO: 212) Primer 2TCT GCA CCG TTA TCA TGA AGT (SEQ ID NO: 213) PDE10A Probe/HEX/TTC CCC TCC /ZEN/TTC TCC TCC CCA /IBFQ/ (SEQ ID NO: 214) Primer 1CTG TTC TTG CCA CTT GAC CA (SEQ ID NO: 215) Primer 2GCT GTA CTC GGA CCT GTT TG (SEQ ID NO: 216) PPP1R1B Probe/HEX/AGG TTC CTC /ZEN/TCC AGG CTC ACT TAG T/IBFQ/ (SEQ ID NO: 217)Primer 1 GGA AAC TCT GAG GAC CAA GTG (SEQ ID NO: 218) Primer 2CTG GGA GAT ACA GGG CTC T (SEQ ID NO: 219) EIF4A2 Probe/6-FAM/AAT GTT GAG /ZEN/CGA GAG GAG TGG AAG C/IBFQ/ (SEQ ID NO: 220)Primer 1 CTG GTG AAG AAG GAA GAA TTG AC (SEQ ID NO: 221) Primer 2TCA AAG TCT CAT ACA AGT CAC AAA G (SEQ ID NO: 222) ATP5B Probe/HEX/TCG GTG CAG /ZEN/GCT ATC TAT GTG CC/IBFQ/ (SEQ ID NO: 223) Primer 1AGG GTC AGT CAG GTC ATC A (SEQ ID NO: 224) Primer 2CAC AAT GCA GGA AAG GAT CAC (SEQ ID NO: 225) UBC Probe/6-FAM/CTC TGA GGC /ZEN/GAA GGA CCA GGT G/IBFQ/ (SEQ ID NO: 226)Primer 1 CAT TCT CTA TGG TGT CAC TGG G (SEQ ID NO: 227) Primer 2AAC ATC CAG AAA GAG TCC ACC (SEQ ID NO: 228) R6/2 Probe/6-FAM/CAG CTC CCT /ZEN/GTC CCG GCG G/IBFQ/ (SEQ ID NO: 229) Primer 1GCT GCA CCG ACC GTG AGT (SEQ ID NO: 230) Primer 2CGC AGG CTG CAG GGT TAC (SEQ ID NO: 231) HttSequence not available from vendor (Life Technologies, Mm01213820_m1)*6-FAM and HEX: 5′ fluorophores; ZEN: internal quencher; IBFQ: 3′quencher

Example 14 Co-Transfection of a Neurotrophic Factor and a HD HttAllele-Specific ZFP TF

The Htt-specific ZFP TFs identified above are co-transfected with ZFPTFs-specific for a brain neurotrophic factor. The ZFP TF specific forbrain neurotrophic factors used are specific for either GDNF or BDNF.

Example 15 Design and Construction of Htt-Targeted Zinc Finger Nucleases(ZFNs)

ZFNs targeting human Htt and mouse Htt were designed to target thesequences flanking the CAG repeats, sequences near the first coding ATG,the stop codon, as well as in early exons. ZFNs were designed andincorporated into plasmids or adenoviral vectors essentially asdescribed in Urnov et al. (2005) Nature 435(7042):646-651, Perez et al(2008) Nature Biotechnology 26(7): 808-816, and U.S. Patent Publication2008/0131962.

Example 16 Cleavage Activity of Htt-Specific ZFNs

To test cleavage activity, plasmids encoding the pairs of humanHtt-specific ZFNs described above were transfected into K562 cells. K562cells were obtained from the American Type Culture Collection and grownas recommended in F-12 medium (Invitrogen) supplemented with 10%qualified fetal calf serum (FCS, Cyclone). Cells were disassociated fromplastic ware using TrypLE Select™ protease (Invitrogen). Fortransfection, one million K562 cells were mixed with 2 μg of thezinc-finger nuclease plasmid and 100 μL Amaxa Solution T. Cells weretransfected in an Amaxa Nucleofector II™ using program U-23 andrecovered into 1.4 mL warm F-12 medium+10% FCS.

Genomic DNA was harvested and a portion of the Htt locus encompassingthe intended cleavage site was PCR amplified. PCR using the AccuprimeHiFi polymerase from InVitrogen was performed as follows: after aninitial 3 minute denaturation at 94° C., 30 cycles of PCR are performedwith a 30 second denaturation step at 94° C. followed by a 30 secondannealing step at 58° C. followed by a 30 second extension step at 68°C. After the completion of 30 cycles, the reaction was incubated at 68°C. for 7 minutes, then at 10° C. indefinitely.

The genomic DNA from the K562 Htt-specific ZFN treated cells wasexamined by the Surveyor™ nuclease (Transgenomic) as described, forexample, in U.S. Patent Publication Nos. 20080015164; 20080131962 and20080159996.

Plasmids encoding the pairs of mouse Htt-specific ZFNs were tested insimilar fashion in Neuro-2a cells.

FIGS. 14A and B show that the ZFNs were capable of targeting the Httgenes with a gene modification efficiency of between 8-40%, assayed asdescribed previously by the amount of indels observed.

Example 17 Targeted Integration of Varying Lengths of TrinucleotideRepeats

The Htt-specific ZFNs with the greatest cleaving activity for sequencesflanking the CAG repeat as described above are used in a targetedintegration strategy to introduce varying lengths of CAG repeat into awild-type copy of Htt. Donors are constructed that contain 50, 80, 109and 180 repeat CAG units. These donors are then transfected into K562cells with plasmids encoding the Htt-specific ZFNs as described above.Verification of donor integration is achieved by genomic DNA isolation,PCR amplification (as described above) followed by sequencing of theregion of interest.

ZFNs identified in the K562 cells which result in targeted integrationof the donor alleles into the Htt allele are used to insert the variablelength donor nucleic acids into human embryonic stem cells (hESC).Successful donor integration is verified by genomic DNA isolation, PCRand sequencing as described above.

Example 18 Disruption/Knock-Out of Wild-Type and/or Mutant Htt

ZFNs that cleave in early exons can result in small insertion ordeletions (in-dels) as a result non-homologous end joining (NHEJ), thiscan generate cell models with one or both alleles of Htt disrupted.

Indicated ZFN pairs were prepared as described above and tested forcleavage activity using the Cel I mismatch as described for Example 8.These ZFN pairs target early exons of human Htt, and thus may be used toknock-out either the wild-type or a mutant Htt allele.

As shown in FIG. 14A, ZFP pairs 29627/29628, 29631/29632 (exon 12) and29637/29638 (exon 18) cleaved the Htt gene and can thus be utilized forgenerating knock-out cell lines.

Example 19 Expression Tagging of Wild-Type and HD Htt Alleles

ZFNs with the greatest cleaving activity for the first or last codingexon are used to tag the wild-type and mutant Htt allele with differentreporter proteins. Donor DNAs for each reporter (A and B) are designedbased on the cleavage site of the lead ZFN pair(s) to allow targetedintegration of the reporter gene to produce an in-frame fusion to Htt.Donor DNAs are co-transfected with the lead ZFN pair(s) into K562 cellsfor selecting the donor DNA construct that gives the highest frequencyof integration.

ZFN pairs were prepared as described above and tested for cleavageactivity using the Cel I mismatch as described for Example 8. The ZFNpairs used target the 3′ end of the Htt coding sequence, and thus may beused to target either a wild-type or a mutant Htt allele. As shown inFIG. 14B, ZFP pairs 25917/25916, 25920/25921 and 25923/25922 werecapable of cleaving the Htt gene and can thus be utilized for theintroduction of a reporter tag.

The selected donor DNA construct for reporter A along with correspondingZFNs are delivered to cells derived from subjects carrying mutant Httgene (e.g. fibroblasts, induced pluripotent cells) Clones are derivedand screened for the target integration of the reporter A. Heterozygousevents are desired and the targeted alleles are identified by PCR.Clones containing a single reporter-tagged Htt allele and unmodified ZFNtarget sequence on the other allele are selected; the donor constructfor reporter B and corresponding ZFNs are transfected to tag the secondallele with the reporter B.

The resulting mouse embryonic stem cell clone contains the wild-type Httallele and mutant allele tagged with two different markers that allowtracking of expression from each allele; these cells are used togenerate mouse models of trinucleotide repeat disorders using standardprotocols.

Example 20 Construction of Active TALE-TF Proteins Against Htt

TALE DNA binding domains were linked to the KRAB repression domain fromthe Kox1 protein (TALE TF) and used to test repression of the Htt genein HD patient (CAG 20/41)-derived fibroblasts. The construction of theTALE proteins was done as described previously (see U.S. Pat. No.8,586,526 and U.S. Patent Publication No. 20130196373, both of which areincorporated herein by reference), and were constructed with threedifferent C-terminal architectures: +63, +231 and +278 as described inU.S. Pat. No. 8,586,526. To construct the TALE TF expression plasmids,the TALEN expression plasmids described previously (see U.S. Pat. No.8,586,526) were used except that the FokI domain used for in the TALENswas replaced with the KRAB repression domain. The linkages of theC-terminus of the TALE protein and the KRAB domain are shown below,where the KRAB domain sequence is indicated by underline. The bold anditalicized text indicates the triple flag tag, the bold text indicatesthe nuclear localization sequence, “[repeats]” indicates the location ofthe TALE repeat unit array (full repeats plus the C-terminal halfrepeat), and the wavy underlined portion shows the sequence of the KRABdomain:

TALE-C63-Kox1:

(SEQ ID NO: 197) M 

 MAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN[repeats]GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPE

TALE-C231-Kox1:

(SEQ ID NO: 198) M 

 MAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN[repeats]GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQR

TALE-C278-Kox1

(SEQ ID NO: 199) M 

 MAPKKKRKVGIHGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN[repeats]GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRAPEQRDALHLPLSWRVKRPRTSIGGGLPDPTPTAADLAASSTVMREQDEDPFAGA

Base recognition was achieved using the canonical RVD-basecorrespondences (the “TALE code”: NI for A, HD for C, NN for G (NK inhalf repeat), NG for T). In some of the TALE TFs, the protein isdesigned to bind the sense (5′-3′) strand of the DNA, while in others,the TALE TF is designed to bind to the anti-sense (3′-5′) strand. Thisset of TALE TFs was designed to target the CAG repeats of the Htt gene.TALE DNA binding proteins often preferentially interact with a ‘T’nucleotide base at the 5′ end of the target, and so since the targetsare CAG repeat regions, it can be predicted that the proteins that bindto the anti-sense DNA strand, and thus CTG repeat sequences with thebase ‘T’ at the 5′ 3nd of the target, might have better binding affinityand specificity and thus repressor activity.

The targets and numeric identifiers for the TALE TFs tested are shownbelow in Table 4. Numeric identifiers are labeled “SBS#”, specificityfor the Sense or Antisense strand is indicated (“S/A”), as well as thetarget, the number of repeat units or RVDs and the type of C-terminus.

TABLE 4 Htt specific TALE-TFs SEQ ID SBS# S/A Target (5′ -3′) NO RVDsC term 102449 S gcAGCAGCAGCAGCAGCAGca 200 17 +63 102450 SgcAGCAGCAGCAGCAGca 201 14 +63 102451 S gcAGCAGCAGCAGca 202 11 +63 102452S gcAGCAGCAGca 203 8 +63 102453 A ctGCTGCTGCTGCTGCTGCtg 204 17 +63102454 A ctGCTGCTGCTGCTGCtg 205 14 +63 102455 A ctGCTGCTGCTGCtg 206 11+63 102456 A ctGCTGCTGCtg 207 8 +63 102457 S gcAGCAGCAGCAGCAGCAGca 20017 +231 102458 S gcAGCAGCAGCAGCAGca 201 14 +231 102459 S gcAGCAGCAGCAGca202 11 +231 102460 S gcAGCAGCAGca 203 8 +231 102462 A ctGCTGCTGCTGCTGCtg205 14 +231 102463 A ctGCTGCTGCTGCtg 206 11 +231 102464 A ctGCTGCTGCtg207 8 +231 102466 S gcAGCAGCAGCAGCAGca 201 14 +278 102467 SgcAGCAGCAGCAGca 202 11 +278 102468 S gcAGCAGCAGca 203 8 +278 102469 ActGCTGCTGCTGCTGCTGCtg 204 17 +278 102470 A ctGCTGCTGCTGCTGCtg 205 14+278 102471 A ctGCTGCTGCTGCtg 206 11 +278 102472 A ctGCTGCTGCtg 207 8+278

The TALE TFs in the table were then tested for Htt repression in HDpatient (CAG 20/41) derived fibroblasts, and the results are shown inFIG. 15. In this experiment, the cells were transfected with either1000, 100 or 10 ng of TALE-TF encoding mRNA. The results for each TALETF assayed are shown in groups of three, representing the threetransfected mRNA amounts. In each grouping, there are also threesamples: the left bar indicates the total Htt expression, the middle barindicates the expression from the CAG20 Htt allele, and the right barindicates the expression from the CAG41 Htt allele. The datademonstrates that there are some TALE TFs that were able to repress bothHtt alleles (see for example 102454), while other TALE TFs were able toselectively inhibit the mutant Htt with the extended CAG repeat (see forexample 102451 and 102472).

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

1. A method of increasing PDE10a, DARPP-32, DRD1 and/or DRD2 levels in a medium spiny neuron (MSN) of a subject by at least 30% relative to a control, the method comprising administering to the subject a genetic repressor of a gene associated with Huntington's disease.
 2. The method of claim 1, wherein the PDE10a, DARPP-32, DRD1 and/or DRD2 levels are increased at least 40% or 50% or more relative to the control.
 3. The method of claim 1, wherein the genetic repressor is a small molecule, a nucleic acid or a protein that inhibits a nucleic acid coding for the huntingtin protein (e.g., genetic DNA or mRNA).
 4. The method of claim 1, wherein the genetic repressor specifically binds to the nucleic acid coding for the huntingtin protein.
 5. The method of claim 1, wherein the genetic repressor comprises an engineered DNA-binding domain.
 6. The method of claim 5, wherein the DNA-binding domain comprises an engineered zinc finger protein, a CRISPR/Cas system or a TAL effector domain.
 7. The method of claim 5, wherein the DNA-binding domain is fused to a functional domain.
 8. The method of claim 7, wherein the functional domain is a transcriptional repression domain or a nuclease.
 9. The method of claim 1, wherein the genetic repressor is administered to the central nervous system (CNS) of a subject.
 10. The method of claim 9, wherein the genetic repressor is administered to the striatum.
 11. The method of claim 1, wherein the genetic repressor is administered using a viral or non-viral vector.
 12. The method of claim 11, wherein the non-viral vector is an adeno-associated virus (AAV) vector.
 13. A method of treating Huntington's Disease in a subject, the method comprising increasing PDE10a, DARPP-32, DRD1 and/or DRD2 levels in a medium spiny neuron (MSN) according to the method of claim
 1. 